Process for the production of polyhydroxyalkanoates

ABSTRACT

Embodiments of the invention relate generally to processes for the production and processing of polyhydroxyalkanoates (PHA) from carbon sources. In several embodiments, PHAs are produced at high efficiencies from carbon-containing gases through the utilization of a regenerative polymerization system.

RELATED APPLICATIONS

This application claims the benefit of Provisional Application Nos.61/237,606, 61/237,609, 61/237,635, 61/237,603, 61/237,616, 61/237,615,61/237,620, 61/237,643, 61/237,633, 61/237,630, 61/237,626, 61/237,642,61/237,639, and 61/237,627, all filed on Aug. 27, 2009, the disclosuresof which are all incorporated by reference herein.

BACKGROUND

1. Field of the Invention

Embodiments of the invention relate to an improved process for theproduction and processing of polyhydroxyalkanoates, and specifically toa process for the production of polyhydroxyalkanoates fromcarbon-containing gases.

2. Description of the Related Art

Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters that serve asenergy storage vehicles in microorganisms. PHAs are biodegradable inboth aerobic and anaerobic conditions, are biocompatible with mammaliantissues, and, as thermoplastics, can be used as alternatives to fossilfuel-based plastics such as polypropylene, polyethylene, andpolystyrene. In comparison to petrochemical-based plastics, which areneither biodegradable nor made from sustainable sources of carbon, PHAplastics afford significant environmental benefits.

The utilization of food crop derived sugars in genetically engineeredmicroorganism-based aqueous fermentation systems is often regarded asthe most efficient and economical platform for PHA production.Specifically, sugar-based PHA production processes are capable ofgenerating high density fermentation cultures and high PHA inclusionconcentrations, and, by maximizing the cell culture density and PHAinclusion concentration therein, it is believed that carbon, chemical,and energy efficiencies are also maximized. For example, comparing a lowcell and PHA concentration process to a high cell and PHA concentrationprocess, a low concentration process requires significantly more, pergiven unit of PHA-containing biomass, i) energy for dewatering cellsprior to PHA extraction treatment, ii) liquid culture volume, andassociated chemicals, mixing energy, and heat removal energy, and iii)both energy and chemicals for separating PHA from biomass. Accordingly,whereas the sugar-based genetically-engineered microorganism PHA processyields maximized cell densities and PHA concentrations relative to lowconcentration processes, it is also regarded as the most carbon,chemical, energy, and, thus, cost efficient PHA production method.

Unfortunately, despite these maximized efficiency advantages,sugar-based PHA production remains many times more expensive than fossilfuel-based plastics production. Thus, given the apparent efficiencymaximization of the high density sugar-derived PHA production process,PHAs are widely considered to be fundamentally unable to compete withfossil fuel-based plastics on energy, chemical, and cost efficiency.

SUMMARY

Despite the environmental advantages of PHAs, the high cost of PHAproduction relative to the low cost of fossil fuel-based plasticsproduction has significantly limited the industrial production andcommercial adoption of PHAs.

To reduce the carbon input cost of the PHA production process,carbon-containing industrial off-gases, such as carbon dioxide, methane,and volatile organic compounds, have been proposed as an alternative tofood crop-based sources of carbon. In addition to the wide availabilityand low cost of carbon-containing gases, carbon-containing gases also donot present the environmental challenges associated with foodcrop-derived sources of carbon. Specifically, whereas food crop-basedcarbon substrates require land, fertilizers, pesticides, and fossilfuels to produce, and also generate greenhouse gas emissions during thecourse of production, carbon-containing off-gases do not require newinputs of land, fertilizers, pesticides, or fossil fuels to generate.Thus, on both an economic and environmental basis, the utilization ofcarbon emissions for the production of PHA would appear to offersignificant advantages over sugar-based PHA production processes.

Unfortunately, the fermentation of carbon-containing gases presentstechnical challenges and stoichiometric limitations that have, in thepast, rendered the gas-to-PHA production process significantly moreenergy and chemical intensive, and thus more costly, than the foodcrop-based PHA production process. Specifically, these technicalchallenges and stoichiometric limitations include: low mass transferrates, low microorganism growth rates, extended polymerization times,low cell densities, high oxygen demand, and low PHA cellular inclusionconcentrations. Whereas sugar-based fermentation systems have theability to generate high cellular densities and PHA inclusionconcentrations, based on cell morphology and mass transfer constraints,carbon-containing gas-based fermentation processes typically generate10-30% of the biomass and intracellular PHA inclusion concentrationsachieved in sugar-based processes. As a result, the ratio ofenergy-to-PHA required to carry out upstream carbon injection, optionaloxygen injection, and culture mixing, as well as downstream PHApurification, significantly exceeds the energy-to-PHA ratio required forsugar-based PHA production methods, thereby rendering theemissions-based process uncompetitive when compared to bothpetroleum-based plastics and sugar-based PHAs.

In light of the potential environmental advantages and carbon costefficiencies of utilizing carbon-containing gases as a source of carbonfor PHA production, there exists a significant need to reduce theenergy, chemical, and carbon input-to-PHA output ratio in a carbonemissions-based PHA production system, and thereby render carbongas-derived PHA economically competitive with petrochemical-basedplastics.

Thus, in several embodiments, the present invention relates to a novelprocess for the conversion of carbon-containing gases into PHAs atpreviously unattainable energy and carbon PHA conversion ratios.

In some embodiments, the invention also relates to a process thatgenerates a carbon emissions-based PHA material that is cost-competitivewith both food crop-based PHAs and fossil fuel-based thermoplastics.

While PHAs are widely considered to be noncompetitive with fossilfuel-based plastics on energy, chemical, and cost efficiency, severalembodiments of the invention relate to a process for producing PHAs fromcarbon-containing gases that yields unexpectedly improved energy,carbon, chemical, and cost efficiencies over sugar-based PHA productionmethodologies.

More specifically, certain embodiments of the invention provide highefficiency, high density, high PHA concentration processes for theproduction of PHA from carbon-containing gases, comprising the steps of:(a) providing a microorganism culture comprising PHA-containing biomass,(b) removing a portion of the PHA-containing biomass from the culture,(c) extracting a portion of PHA from the removed culture to produceisolated PHA and PHA-reduced biomass, (d) purifying the isolated PHA,and (e) returning the PHA-reduced biomass to the culture to cause theculture to convert the carbon within the PHA-reduced biomass into PHA.In several embodiments, carbon output from the system is wholly orsubstantially only in the form of PHA.

In several embodiments, a system for using a microorganism culture toconvert a carbon-containing gas into PHA at high efficiencies isprovided. Microorganisms are cultured using a combination of one or morecarbon-containing gases and PHA-reduced biomass, or derivatives thereof,as sources of carbon to produce PHA-containing biomass. A portion of thePHA-containing biomass is then removed from the culture, and PHA isextracted from the removed PHA-containing biomass to createsubstantially PHA-reduced biomass and substantially isolated PHA.

Typically, PHA is present in the PHA-containing biomass of gas-utilizingmicroorganisms at concentrations in the range of about 5%-60%, andapproximately 40-95% of the PHA-containing biomass is discarded from thesystem following PHA extraction. In some cases, PHA is present ingas-utilization microorganisms in the range of about 1-90%, including atabout 1%, 3%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70%, 80%, or 90%, and approximately 10-99% of the PHA-containing biomassis discarded from the system following PHA extraction, including 99%,97%, 95%, 93%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%,20%, or 10% of the PHA-containing biomass. Rather than discarding theremaining, e.g., 40-95% of the PHA-reduced biomass, in one embodiment ofthe invention, the PHA-reduced biomass is returned back to themicroorganism culture to be regenerated as PHA by a microorganismculture capable of utilizing PHA-reduced biomass, or a derivativethereof, as a source of carbon for PHA production, thereby creating aregenerative closed-loop polymerization system. By using PHA-reducedbiomass as a source of carbon for PHA production in microorganismsgrowing as or in association with gas-utilizing microorganisms, PHA canbe produced from carbon-containing gases at surprisingly andunexpectedly improved carbon, energy, and chemical efficiencies, sincecarbon from carbon-containing gases that would otherwise be discarded isregenerated as PHA in a microorganism culture, and microorganisms thatproduce PHA from carbon-containing gases at low concentrations (e.g.,5-60% PHA by weight, or less than 70% PHA by weight) can, in someembodiments, be utilized to produce PHA at significantly increasedcarbon-to-PHA efficiencies. In some embodiments, the regeneration stepis repeated to form an essentially closed-loop system. Thus, in someembodiments, the carbon output from the system is at least 1%, 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% PHA In other words,at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 70%, 80%, 90%, 95%, or 99% ofthe carbon entering the system is converted into PHA. In otherembodiments, 1-5%, 5-10%, 10-20%, 20-30%, 30%-40%, 40%-50%, 50%-60%,60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-99% (and overlapping rangesthereof) of the carbon entering the system is converted to PHA. Byregenerating PHA-reduced biomass as PHA in a microorganism culture, thepercentage of carbon from a carbon containing gas that is converted toPHA is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, or greater than systems that do not employ theregenerative or closed-loop system disclosed herein. In someembodiments, the regeneration (e.g. return and/or recycling of thePHA-reduced biomass) step is repeated at least 2, 3, 4, 5, 6, 7, 8, 9,or 10 (or more) times. In some embodiments, the regeneration step isrepeated until at least 90% to 95% of the carbon input into the systemis converted into PHA. In some embodiments, the regeneration step isrepeated as many times as desired to reach a particular percentageconversion of carbon to PHA.

Several embodiments of the invention provide for the production of PHAfrom carbon-containing gases at previously unattainable energy, carbon,and chemical efficiencies by way of providing a microorganism culturecapable of metabolizing the carbon within both a carbon-containing gasand PHA-reduced biomass, manipulating the conditions of the culture tocause the culture to produce PHA, removing a portion of PHA-containingbiomass from the culture, extracting the PHA within the removedPHA-containing biomass to create substantially isolated PHA andsubstantially PHA-reduced biomass, returning the PHA-reduced biomass tothe culture and contacting the PHA-reduced biomass with the culture tocause the culture to metabolize the carbon within the PHA-reducedbiomass into PHA, and purifying the isolated PHA. Thus, an advantage ofseveral embodiments of the invention is the production of PHA fromcarbon-containing gases at significantly improved energy, carbon, andchemical efficiencies.

The process according to several embodiments disclosed herein yields arange of surprising benefits over current gas-based PHA productiontechnologies. To begin, whereas the cell density of gas-basedfermentation processes is traditionally limited by the mass transfer ordiffusion rates of one or more factors, such as light, oxygen, carbondioxide, methane, or volatile organic compounds, several embodimentsdisclosed herein enable the generation of cell densities thatsignificantly exceed cell densities attainable in the current practice(e.g., by more than 1%, 10%, 20%, 30%, 50%, 80%, 100% or more), andthereby enables cost-efficient system mixing, aeration, heat control,and dewatering. For example, current methane-based PHA productionsystems are known to be capable (based on cell morphology and masstransfer characteristics) of generating approximately 60 g/L of biomasswith an overall PHA concentration of 55%, or 33 g/L PHA. In contrast, inseveral embodiments of the invention, cell densities of approximately135 g/L with an overall PHA concentration of 70%, or 94.5 g/L PHA aregenerated in a methane-based PHA production system. In some embodiments,cell densities of approximately 10 g/L, 20 g/L, 30 g/L, 60g/L, 75 g/L,100g/L, 125g/L, 135 g/L, 150g/L or greater are achieved. In someembodiments, overall PHA concentration in such cultures ranges fromapproximately 1% to 20%, 20% to 30%, 30% to 55%, 55% to 60%, 65% to 70%,70% to 80%, and overlapping ranges thereof result. In severalembodiments, such PHA concentration ranges represent significant,unexpected, and surprising improvements over traditional processes,e.g., processes that are limited to low cell densities and/or PHAconcentrations.

As an non-limiting example of the impact of this improvement on energyefficiency, the energy required, on an energy input-to-PHA output basis,to aerate, mix, and dewater a 135 g/L solution with a PHA concentrationof 70% by weight is 186% less than the energy required to aerate, mix,and dewater a 60 g/L microorganism solution comprising 40% PHA byweight. It shall be appreciated that variations in the energy efficiencygains based on the systems and processes disclosed herein may occur,depending on the culture conditions, the strain or organisms used, andthe initial gas stream or other carbon source. In several embodiments,even modest increases in efficiency have substantial benefits. Forexample, the ability to efficiently use an input gas having a low carbonconcentration that would not otherwise be useful in PHA production mayprevent the release of such a gas into the environment and/or reactionof the gas with other atmospheric compounds, thereby reducing theadverse impact of the low carbon concentration gas on the environment(e.g., destruction of ozone, greenhouse gas emission, pollution, etc.).

Additionally, whereas current gas-based PHA production systems producesignificant carbon losses as a result of the low PHA inclusionconcentrations of gas-utilizing microorganisms (i.e., a significantportion of carbon and energy input is lost as biomass), severalembodiments of the invention enable the generation of overall carboninput yield efficiencies approaching maximum substrate values; e.g.,100% carbon input-to-PHA yield, minus respiration and/or downstreamprocessing losses. In some embodiments, at least 5%, at least 10%, atleast 30%, at least 50%, at least 70%, or at least 90%, carboninput-to-PHA yield is achieved. It is one important advantage of severalembodiments of the invention that maximum carbon yield efficiencies areunexpectedly and surprisingly generated in a PHA production systememploying gas-utilizing microorganisms, and particularly, in someembodiments, in PHA production systems employing gas-utilizingmicroorganisms that produce low biomass and/or PHA inclusion densities.

In some embodiments, the microorganism culture is a mixed culture,comprising heterotrophic microorganisms, methanotrophic microorganisms,autotrophic microorganisms, bacteria, yeast, fungi, algae, orcombinations thereof. In other embodiments, the microorganism culturemay be one or more cultures (e.g., a plurality of cultures). In someembodiments, the cultures are grown in one or more bioreactors. In someembodiments, the bioreactors utilize one or more culture conditions,including both aerobic and anaerobic conditions. In some embodiments,the microorganism culture converts PHA-reduced biomass to methane in ananaerobic process and subsequently to PHA in an aerobic process, suchthat PHA-reduced biomass is first anaerobically metabolized to methaneand then used as methane to produce biomass and PHA in a methanotrophicculture.

In several embodiments, at least part of the microorganism culture is amixed culture capable of metabolizing carbon-containing gases, includingmethane, carbon dioxide, greenhouse gases, and/or various other volatileorganic compounds, into biomass and/or PHA. In some embodiments, themicroorganism culture comprises a two phase system of anaerobic andanaerobic metabolism, whereby carbon-containing gas is produced in afirst substantially anaerobic phase and subsequently converted into PHAin a second phase, wherein the microorganism culture in the first phaseis substantially anaerobic and the culture in the second phase is eitheranaerobic or aerobic, wherein the two phases may be operated in onesingle vessel or in multiple vessels.

In some embodiments, at least one or more of the microorganisms arecontacted with artificial and/or natural light during one or more stepsof the methods disclosed herein.

In some embodiments, at least one of more of the microorganisms iscontacted with dissolved oxygen during one or more steps of the methodsdisclosed herein.

In some embodiments, at least one of more of the microorganisms iscultured at atmospheric, sub-atmospheric, or above-atmosphericpressures.

In some embodiments, at least one of more of the microorganisms canutilize only a carbon-containing gas as a source of carbon.

In several embodiments, at least one of more of the microorganisms canutilize carbon derived from a PHA-reduced biomass as a source of carbon.In other embodiments, at least one or more of the microorganisms is aheterotrophic microorganism capable of converting PHA-reduced biomassinto, carbon dioxide, oxygen, biomass, and/or PHA.

In several embodiments, at least one or more of the microorganisms arecultured using carbon derived from both a carbon-containing gas and aPHA-reduced biomass.

In some embodiments, the microorganism culture is a pure culture. Insome embodiments, the cultures are maintained in semi-sterile or sterileconditions.

In some embodiments, the microorganism culture is a mixed, non-sterileculture, including a naturally equilibrating consortium ofmicroorganisms.

In several embodiments, the microorganism culture is at least partiallycomprised of genetically engineered microorganisms.

In some embodiments, the microorganism culture is a mixed culturecomprising a combination of naturally occurring and geneticallyengineered microorganisms.

In several embodiments, the PHA is removed from the microorganismculture by solvent extraction, including solvent extraction attemperatures ranging from 0° C. to 200° C. and at pressures ranging from−30 psi to 200 psi.

In several embodiments, the PHA is removed from the microorganismculture through the utilization of ketones, alcohols, and/or chlorinatedsolvents.

In several embodiments, the PHA is removed from the microorganismculture by hypochlorite digestion and/or chlorine-based solventextraction.

In several embodiments, the PHA is removed from the microorganismculture by supercritical carbon dioxide extraction.

In several embodiments, the PHA is removed from the microorganismculture by protonic non-PHA cell material dissolution.

In several embodiments, the PHA is partially removed from themicroorganism culture to create a PHA-rich phase and a PHA-poor phase.

In several embodiments, the PHA is removed from the microorganismculture to render the PHA substantially free of non-PHA material,including substantially 5%, 10%, 20%, 30%-40%, 40%-50%, 50%-60%,60%-70%, 70%-80%, 80-90%, 90-99% or more pure PHA by weight.

In several embodiments, the PHA is removed from the microorganismculture by manipulating the pH of the microorganism culture.

In certain embodiments, at least one of more of the microorganisms arecontacted with methane, carbon dioxide, oxygen, and/or a combinationthereof.

In several embodiments, multiple culture vessels are employed, such thatmicroorganism growth, PHA synthesis, PHA-reduced biomass metabolism, andPHA removal are carried out in separate vessels.

In other embodiments, microorganism growth, PHA-reduced biomassmetabolism, and PHA synthesis occurs in a single vessel.

In still other embodiments, microorganism growth and PHA synthesis occurin a single vessel and PHA extraction is carried out in one or moreseparate vessels.

In several embodiments of the process as disclosed herein, PHA synthesisis regulated by manipulating the concentration of a material in theprocess, wherein the material is oxygen, methane, carbon dioxide,nitrogen, phosphorus. copper, iron, manganese, carbon, magnesium,potassium, cobalt, aluminum, sulfate, chlorine, boron, citric acid, orEDTA.

In several embodiments, the microorganism culture comprises one or morestrains of microorganisms collectively capable of converting the carbonwithin a carbon-containing gas into cellular biomass and the carbon fromcellular biomass or methane into PHA.

In several embodiments, the microorganisms are subjected to filtration,centrifugation, settling, and/or density separation.

In several embodiments, the isolated PHA and/or the PHA-reduced biomassis subjected to filtration, centrifugation, settling, and/or densityseparation.

In some embodiments, the process further comprises washing the recoveredPHA with water, solvent, or other liquid-based agents to purify the PHA.

In several embodiments, the process further comprises oxidizing therecovered PHA to purify the PHA.

In several embodiments, the process further comprises drying therecovered PHA to remove volatiles such as water and/or one or moresolvents.

In several embodiments of the invention, methods for the production ofPHA are provided. In one embodiment, the method comprises: (a) providinga microorganism culture comprising PHA-containing biomass, (b) removinga portion of the PHA-containing biomass from the culture, (c) extractinga portion of the PHA from the removed PHA-containing biomass to produceisolated PHA and PHA-reduced biomass, (d) returning the PHA-reducedbiomass to the culture to cause the culture to convert the carbon withinthe PHA-reduced biomass into PHA, and (e) purifying the isolated PHA.

In one embodiment, the microorganism culture utilizes the PHA-reducedbiomass, or derivatives thereof, such as carbon dioxide, methane, orvolatile organic acids, volatile fatty acids, volatile organiccompounds, non-methane organic compounds, and one or morecarbon-containing gas as a source of carbon. In one embodiment, the gasis selected from the group consisting of methane, carbon dioxide,volatile organic compounds, and hydrocarbons. In one embodiment, the gasis derived from one or more sources from the group consisting of:landfills, wastewater treatment plants, power production facilities orequipment, agricultural digesters, oil refineries, natural gasrefineries, cement production facilities, and/or anaerobic organic wastedigesters.

In some embodiments, the carbon in the PHA-reduced biomass is derivedfrom one or more gases from the group consisting of methane, biogas,carbon dioxide, volatile organic compounds, natural gas, wastewatertreatment methane and VOCs, and hydrocarbons.

In some embodiments, natural and/or artificial light is utilized toinduce the metabolism of the carbon dioxide by the culture.

In some embodiments, the microorganism culture comprises one strain, ora consortium of strains, of microorganisms, including one or moremicroorganisms selected from the group consisting of bacteria, fungi,yeast, and algae, and combinations thereof.

In some embodiments, the microorganism culture comprises one or moremicroorganisms from the group consisting of: methanotrophicmicroorganisms, carbon-dioxide utilizing microorganisms, anaerobicmicroorganisms, methanogenic microorganisms, acidogenic microorganisms,acetogenic microorganisms, heterotrophic microorganisms, autotrophicmicroorganisms, cyanobacteria, and biomass-utilizing microorganisms, andcombinations thereof.

In some embodiments, at least a portion of the microorganism culture isnaturally occurring. In some embodiments, at least a portion of themicroorganism culture is and/or genetically engineered. In someembodiments, naturally occurring and genetically engineeredmicroorganisms are both used in the culture.

In some embodiments, the microorganism culture is at least partiallymaintained under above-atmospheric pressure.

In some embodiments, the PHA-containing biomass includes one or moremicroorganism-derived materials selected from the group consisting of:intracellular, cellular, and/or extracellular material, including apolymer, amino acid, nucleic acid, carbohydrate. lipid, sugar,polyhydroxyalkanoate, chemical, and/or metabolic derivative,intermediary, and/or end-product. In some embodiments, thePHA-containing biomass includes one or more microorganism-derivedmaterials selected from the group consisting of: methane, volatileorganic compounds, carbon dioxide, and organic acids.

In one embodiment, the PHA-containing biomass contains less than about95% water, including less than about 90%, 85%, 80%, 75%, or 70% water.

In some embodiments, the PHA-containing biomass is mixed with achemical, including one or more chemicals from the group consisting of:methylene chloride, acetone, ethanol, methanol, ketones, alcohols,chloroform, and dichloroethane, or combinations thereof.

In one embodiment, the PHA-containing biomass is processed throughhomogenization, heat treatment, pH treatment, enzyme treatment, solventtreatment, spray drying, freeze drying, sonication, and microwavetreatment, or combinations thereof.

In one embodiment, the PHA-reduced biomass includes the PHA-containingbiomass wherein at least a portion of the PHA has been removed from thePHA-containing biomass. In another embodiment, the PHA-reduced biomassincludes methane, carbon dioxide, and organic compounds produced fromthe PHA-reduced biomass.

In some embodiments, the PHA-reduced biomass is subject to dewatering,chemical treatment, sonication, additional PHA extraction,homogenization, sonication, heat treatment, pH treatment, hypochloritetreatment, microwave treatment, microbiological treatment, includingboth aerobic and anaerobic digestion, solvent treatment, water washing,solvent washing, and/or drying, including simple or fractionaldistillation, spray drying, freeze drying, and/or oven drying, orcombinations thereof.

In several embodiments, the microorganism culture is maintained in asterile, semi-sterile, or non-sterile environment.

In one embodiment, the PHA includes one or more PHA selected from thegroup consisting of: polyhydroxybutyrate (MB), polyhydroxyvalerate(PHV), polyhydroxybutyrate-covalerate (PHB/V), polyhydroxyhexanoate(PHHx), and short chain length (SCL), medium chain length (MCL), andlong chain length (LCL) PHAs.

In several embodiments, the metabolism, growth, reproduction, and/or PHAsynthesis of the culture is controlled, manipulated, and/or affected bya growth medium. In some embodiments, the bioavailable and/or totalconcentration of nutrients within the growth medium, such as copper,iron, oxygen, methane, carbon dioxide, nitrogen, magnesium, potassium,calcium, phosphorus, EDTA, calcium, sodium, boron, zinc, aluminum,nickel, sulfur, manganese, chlorine, chromium, molybdenum, and/orcombinations thereof are manipulated (e.g., increased, decreased, ormaintained) in order to control the metabolism, growth, reproduction,and/or PHA synthesis of the culture In some embodiments, a singlenutrient in the growth medium is manipulated, while in some embodiments,more than one nutrient in the growth medium is manipulated to achievethe desired effect on the culture.

In one embodiment, the conversion of the PHA-reduced biomass into thePHA is induced and/or controlled by manipulating the composition of themedium. As discussed herein, the conversion of PHA-reduced biomass intothe PHA can be controlled in a time-dependent manner to maximize theefficiency of conversion. In some embodiments, conversion to PHAproduction is induced about 1-12 hours, about 5-15 hours, or about 8-24hours after PHA-reduced biomass is re-introduced into the culture. Insome embodiments, longer times, e.g., about 24 hours to several days orweeks, are employed.

In one embodiment, the conversion of the PHA-reduced biomass into thePHA is effected by manipulating the concentration one or more elementsselected from the group consisting of: nitrogen, methane, carbondioxide, phosphorus, oxygen, magnesium, potassium, iron, copper,sulfate, manganese, calcium, chlorine, boron, zinc, aluminum, nickel,and/or sodium, and combinations thereof.

In some embodiments, the PHA is at least partially removed from thePHA-containing biomass using one or more extraction agents selected fromthe group consisting of: solvents, including methylene chloride,acetone, ethanol, methanol, or dichloroethane, supercritical carbondioxide, sonication, homogenization, water, heat, distillation, spraydrying, freeze drying, enzymes, surfactants, acids, bases, hypochlorite,peroxides, bleaches, ozone, EDTA, and/or combinations thereof.

In one embodiment, the extraction process is substantially carried outat intracellular temperatures less than 100° C. In other embodiments,temperatures for extraction range from about 10° C. to 30° C., fromabout 30° C. to 50° C., from about 50° C. to 70° C., from about 70° C.to 90° C., from about 90° C. to about 120° C., or higher. In anotherembodiment, cells are reused for polymerization following the extractionprocess as viable cells.

In one embodiment, the removal of the PHA from the culture causes theculture to be temporarily deactivated, such that the culture, orelements thereof, may be further used for the synthesis of PHA. Incertain embodiments, deactivation is beneficial because it allows forthe delay of PHA production, transfer of material to another productionarea, and the like. In some embodiments, deactivation allows a tailoredPHA production time frame. In some embodiments, the reuse of cells forpolymerization is beneficial because it avoids or reduces the need toproduce new biomass prior to polymerization, thereby reducing thecarbon, chemical, and energy requirement of PHA production.

In one embodiment, a PHA produced according to the several embodimentsdescribed herein is provided.

In several embodiments, processes for the production of PHA from acarbon-containing gas are provided. In one embodiment, the processcomprises the steps of: a) providing a growth medium comprising amicroorganism culture capable of utilizing the carbon within one or morecarbon-containing gas and PHA-reduced biomass, b) manipulating themedium to cause the culture to produce PHA, c) removing at least aportion of the PHA within the culture to create substantially isolatedPHA and substantially PHA-reduced biomass, d) purifying the isolatedPHA, and e) returning the PHA-reduced biomass to the culture to causethe culture to metabolize the carbon within the PHA-reduced biomass intoPHA.

In one embodiment, the carbon-containing gas is selected from the groupconsisting of: methane, carbon dioxide, toluene, xylene, butane, ethane,methylene chloride, acetone, ethanol, propane, methanol, vinyl chloride,volatile organic compounds, hydrocarbons, and combinations thereof.

In one embodiment, the invention comprises a PHA comprising carbonderived from PHA-reduced biomass, wherein the PHA-reduced biomasscomprises carbon derived from one or more carbon-containing gas.

In one embodiment, a PHA derived from a carbon-containing, greenhousegas, including methane, carbon dioxide. or combinations thereof, isprovided. In some embodiments, use of such a gas is particularlyadvantageous, as it allows for the simultaneous production of PHA atlower energy costs and higher efficiencies, but also removes a portionof a destructive gas from the atmosphere. In some embodiments, processesand systems as disclosed herein are particularly well suited for usenear sources of such gases (e.g., landfills, power production plants,anaerobic digesters, etc.) for onsite conversion of harmful gasses to acommercially valuable product.

In several embodiments, processes for the oxidation of methane areprovided. In one embodiment, the process comprises: providing a cultureof methanotrophic and autotrophic microorganisms, providing a growthculture medium comprising dissolved methane and carbon dioxide, andcontacting the culture with light to cause the culture to convert thecarbon dioxide into oxygen, whereby the culture utilizes the oxygen tooxidize the methane, thereby reducing or eliminating the need for anextraneous source of oxygen to drive methanotrophic metabolism.

In some embodiments, the light used to contact the culture is artificiallight. In some embodiments, the light used to contact the culture isnatural light. In other embodiments, combinations of natural andartificial light are used. In some such embodiments, wavelengths ofartificial light are specifically filtered out or controlled such thatthe culture is exposed to a broader or more controlled overall spectrumof light (e.g., the sum of wavelengths of natural light and artificiallight). In some embodiments, the source of light also functions togenerate heat, which can be used to maintain optimal culturetemperatures. In other embodiments, light input is regulated by time,such that specific cultures of autotrophic and/or heterotrophicmicroorganisms are selected for or optimized according to the durationand/or pattern of light injection (e.g., 0-12 or 12-24 hours lightinjection, 0-12 or 12-24 hours dark incubation, multi-second pulsation,etc.).

In one embodiment, the addition of light reduces the need for exogenousoxygen sources. While such embodiments provide an advantage in reducingcosts of input materials, in some embodiments, an exogenous source ofoxygen, including air, is added to the culture.

In some embodiments the addition of autotrophic microorganisms to theculture impacts the metabolism of the culture. In such an embodiment,the timed and planned addition, activation, or metabolic enhancement ofautotrophic organisms can be based on the desire for changing the rateof methane oxidation.

In some embodiments, a system is used for PHA production comprisingproviding i) a culture of autotrophic, methanotrophic, methanogenic,and/or heterotrophic microorganisms and ii) a first gas comprisingcarbon dioxide, methane, volatile organic compounds, oxygen, and/orother gas, whereby the culture of microorganisms are caused to used thefirst gas to generate a second gas comprising carbon dioxide, methane,oxygen, volatile organic compounds, and/or other gas, whereby theculture subsequently is caused to utilize the second gas for thegeneration of PHA, which can then be isolated and purified according toseveral embodiments disclosed herein. In some embodiments, the first gascan be methane, carbon dioxide, oxygen, or volatile organic compounds.In other embodiments, the second gas can be oxygen, methane, carbondioxide, or other volatile organic compounds. In some embodiments,microorganisms can be used to convert carbon dioxide to biomass whichcan in turn be used to produce methane, which can be subsequently usedto produce PHA. In other embodiments, microorganisms can be used toconvert carbon dioxide to oxygen which can in turn be used to producePHA. As disclosed herein, the products generated at each of these stepsmay be recycled (e.g., splitting a portion of the autotrophic cultureand recycling it to generate additional biomass, generating reduced-PHAbiomass and recycling it into the methanotrophic culture to generateadditional biomass and additional PHA).

In several embodiments, processes for producing autotrophicmicroorganisms using only methane as a carbon input are provided. In oneembodiment, the process comprises: adding methane, oxygen, andmethane-utilizing microorganisms to a culture of autotrophicmicroorganisms, whereby the methane-utilizing microorganisms convert themethane into carbon dioxide, and whereby the autotrophic microorganismsutilize the carbon dioxide as a source of carbon, thereby reducing oreliminating the need for an extraneous source of carbon dioxide to driveautotrophic metabolism. In some embodiments, addition of methanotrophicand/or heterotrophic microorganisms to the culture impacts themetabolism of the autotrophic microorganisms. As discussed herein, thepurposeful addition of such microorganisms at particular times allowsfor specific levels of control over the overall output and operation ofthe system.

In several embodiments, processes for oxidizing methane at lowconcentrations are provided. In one embodiment, the process comprises:culturing methanotrophic microorganisms in a medium comprising water,dissolved methane, dissolved oxygen, and mineral salts, adding methanolto the medium at a rate and volume sufficient to cause themicroorganisms to reduce the concentration of the methane in the medium,whereby substantially all of the methane within the medium is utilized,thereby enabling methanotrophic microorganisms to metabolize methanepresent at low bioavailable concentrations. In some embodiments, gascontaining less than 20% methane by volume is contacted with the medium.In some embodiments, gas containing less than 1% methane by volume iscontacted with the medium. In some embodiments, the methanol is producedby microorganism metabolism.

In several embodiments, processes for separating water frommicroorganism biomass are provided. In one embodiment, the processcomprises: providing biomass mixed with water in a liquid medium, mixingthe medium with a liquid agent selected from the group consisting ofketones, alcohols, chlorinated solvents, derivatives thereof, orcombinations thereof, and subjecting the mixture to a filtration step.In several embodiments, this enables the efficient separation of biomassfrom water. In some embodiments, such methods reduce or eliminate theneed for centrifugation in the separation process. In some embodiments,the liquid agent is acetone, ethanol, isopropanol, and/or methanol. Inother embodiments, other liquid agents that are miscible with water areused to separate the biomass from the aqueous portion of the mixture. Inseveral embodiments separation is achieved by centrifugation(high-speed, low-speed), gravity separation, multi-stage filtration, orcombinations thereof.

In several embodiments, processes for extracting a polyhydroxyalkanoatefrom a PHA-containing biomass are provided. In one embodiment, theprocess comprises the steps of: (a) providing a PHA-containing biomasscomprising PHA and water, (b) mixing said biomass with a solvent at atemperature sufficient to dissolve at least a portion of said PHA intosaid solvent and at a pressure sufficient to enable substantially all orpart of said solvent to remain in liquid phase, thereby producing aPHA-lean biomass phase and a PHA-rich solvent phase comprising water,PHA and solvent (c) separating said PHA-rich solvent phase from saidPHA-lean biomass phase at a temperature and pressure sufficient toenable substantially all or part of said solvent to remain in liquidphase and prevent substantially all or part of said PHA within saidPHA-rich solvent phase from precipitating into said water, (d) reducingthe pressure or increasing the temperature of said PHA-rich solventphase to cause said PHA-rich solvent to vaporize and said PHA toprecipitate or otherwise become a solid PHA material while maintainingthe temperature and/or pressure of the PHA-rich solvent phase to preventall or part of the temperature-dependent precipitation of said PHA intosaid water, and (e) collecting said solid PHA material, includingoptionally separating said solid PHA material from said solvent and/orsaid water.

In some embodiments, suitable solvents include acetone, ethanol,methanol, dichloroethane, and/or methylene chloride. Depending on thesolvent selected, in some embodiments, separating the solid PHA materialfrom solvent and/or water is achieved by increasing the temperature ofthe mixture. In other embodiments, separation is achieved throughreducing the pressure of the solvent, PHA, and/or water. In someembodiments, combinations of temperature changes and pressure changesare used to optimally separate solid PHA material from solvent and/orwater. In some embodiments, evaporation of solvent and/or water occursin a rapid fashion, thereby reducing the need for temperature orpressure changes. Advantageously, certain embodiments of the processesdisclosed herein may optionally be carried out in a batch,semi-continuous, or continuous manner. Thus, the process can be tailoredto the needs of the producer at any given time.

In several embodiments, processes for modifying the functionalcharacteristics of a PHA are provided. In one embodiment, the processcomprises providing a first PHA and a second PHA, wherein the molecularweight of said second PHA is greater than the molecular weight of saidfirst PHA, and combining said first PHA with said second PHA to modifythe functional characteristics of both said first PHA and second PHA. Insome embodiments, both the first PHA and the second PHA are PHB, and insome embodiments, one or more of the first and second PHA comprisesPHB/V. In one embodiment the first PHA and the second PHA is PHB orPHBV.

In some embodiments, the molecular weight of the first PHA is greaterthan about 500,000 Daltons and the molecular weight of the second PHA isless than about 500,000 Daltons. However, in some embodiments, themolecular weight can be adjusted. For example, in some embodiments, afirst PHA is subjected to a temperature sufficient to reduce themolecular weight of the first PHA. Thereafter, it can be combined withthe second PHA. It shall be appreciated that the second PHA could alsooptionally be exposed to temperature in order to adjust its molecularweight. In some embodiments, the molecular weight of the second PHA isgreater than about 800,000 Daltons. In certain embodiments, themolecular weight of said second PHA is greater than about 1,000,000Daltons. In some embodiments, the molecular weights of the first andsecond PHA are specifically tailored relative to one another, (e.g., aratio of 1:2, 1:4, 1:6, 1:8, 1:10, etc.) in order to maximize thealterations in functional characteristics.

In several embodiments, processes for increasing the penetration depthof light in a liquid are provided. In one embodiment, the processcomprises the steps of (a) directing light into a liquid medium in theform of a light path and (b) reducing the density of liquid in the lightpath.

In several embodiments, the density of the liquid in the light path isreduced by adding gas to said liquid in the light path. In someembodiments, the gas is air, oxygen, methane, carbon dioxide, nitrogen,and/or a combination thereof. In some embodiments, the gas issimultaneously added along with the light. In certain embodiments, thegas and the light are emitted or injected into the liquid through acommon material, such as a permeable or semi-permeable membrane throughwhich light and/or gas traverse. In other embodiments, the light and thegas are added separately. In such embodiments, customization of theaddition is possible. For example, gas can be added in pulses (e.g.,on/off sequences), continuously, or in bracketed time frames around theaddition of light. In some embodiments, the addition of light and gasare coordinated to maximize the penetration of the light. For example aburst of gas followed by a burst of light (or overlapping to somedegree) may advantageously increase the penetration of the light.

In several embodiments, processes for modifying the pH in amicroorganism culture medium are provided. In one embodiment, theprocess comprises the steps of: (a) providing a culture mediumcomprising water and microorganisms, (b) adding a first source ofnitrogen to the medium to cause the microorganisms to metabolize thenitrogen and thereby increase the concentration of either hydroxyl ionsor protons, respectively, in the medium, and (c) adding a second sourceof nitrogen to the medium to cause the microorganisms to metabolize thenitrogen and thereby increase the concentration of either protons orhydroxyl ions. respectively. in the medium. In other embodiments. asource of nitrogen is added to the culture that increases the pH of themedium, wherein the metabolism of the nitrogen source causes the pH ofthe medium to decrease, thereby reducing or eliminating the need for anadditional pH adjustment step.

In several embodiments, low shear processes for adding gas to amicroorganism culture medium are provided. In one embodiment, theprocess comprises the steps of: (a) providing a liquid medium and a gas,(b) contacting the medium with the gas in a first container to cause atleast a portion of the gas to dissolve in the medium, (c) providing asecond container, and (d) transferring at least a portion of the liquidcomprising the gas within the first container to the second container.In some embodiments, a mixer is also provided, in order to dissolve aportion of the gas in the medium. In some embodiments, the mixer is apump or agitator or high shear mixer. In some embodiments, the mixercomprises a centrifugal pump. In still other embodiments, the gas itselfprovides a mixing function. For example, the injection of gas into amedium will result in gas bubbles, which, if released at the bottom of acontainer comprising medium, will not only promote the dissolution ofgas into the medium, but mix the medium as the bubbles rise.

In several embodiments, processes for injecting gas into a pressurizedmicroorganism culture vessel are provided. In one embodiment, theprocess comprises the steps of: providing a vessel comprising a mediumcomprising microorganisms, adding a gas into the vessel that can bemetabolized by the microorganisms, and adjusting the flow rate of thegas into the vessel according to the rate of change of pressure withinthe vessel. In some embodiments, the gas is oxygen, methane, carbondioxide, or combinations thereof. Choice of the gas depends on thevessel used and the culture within the vessel. In some embodiments,backpressure monitoring allows for optimal gas injection for a givenculture (e.g., if certain cultures react more quickly to administrationof a gas and rapidly increase pressure, flow can be coordinatelyreduced).

In one embodiment, a process for producing light in a liquid medium isprovided. In one embodiment, the process comprises the steps of a)providing a liquid, b) providing a light-emitting unit or materialcomprising two conductive leads and a light-emitting conjuncture betweenthe conductive leads, or a material that will emit light when contactedwith electrons and c) inducing a voltage in the liquid, thereby inducingthe movement of electrons in the conductive leads of the light-emittingunit or inducing electrons to contact the material, thereby causing thelight-emitting unit or material to emit light In some embodiments, thematerial is a phosphor, phosphoric, and/or luminescent material,including an electroluminescent phosphor.

In one embodiment, AC voltage is induced in a liquid by inserting thetwo leads of a 115V AC power source into a liquid. Without being boundby theory, it is believed that a liquid carrying an AC voltage iscapable of inducing the movement of electrons into and through alight-emitting device suspended in the liquid and not contacting the two115V AC power source leads due to the oscillating nature of electrons inan AC circuit, such that AC voltage in a liquid causes electrons to fillconductive paths connected to the liquid, in spite of the resistance ofthe conductive paths relative to the liquid, and will oscillate as an ACcurrent in those conductive paths, thereby performing work, e.g.,generating light in a light emitting diode. As a non-limiting example,light is produced in a liquid medium by a) placing a light-emittingdiode in a liquid comprising water and electrolytic ions, and b)inducing an AC voltage in the liquid, wherein c) the induction of ACvoltage in the electrolytic liquid causes the light-emitting diode togenerate light.

Through experimentation, Applicant unexpectedly discovered that one ormultiple light emitting units will emit light in a liquid when ACvoltage is applied to the liquid and when the light-emitting units arerated for voltages and amp draws commensurate with available electricalenergy. The production of autotrophic microorganisms is fundamentallyconstrained by the ability of light to penetrate through a liquid andthereby enable photosynthesis. Prior to Applicant's invention asdisclosed herein, no methods were believed to be known to produce lightin a liquid through the utilization of light-emitting devices physicallyunconnected to an electrical voltage source vis-à-vis solid conductivematerial. In one embodiment, the utilization of one or more, andpreferably many, free-floating light-emitting units in an electricallycharged liquid comprising autotrophic microorganisms enables a very highlight transmission efficiency, wherein previous light penetrationconstraints are largely overcome and high autotrophic microorganismdensities are fully enabled.

In several embodiments, a method for producing a polyhydroxyalkanoate(PHA) in a microorganism culture is provided. In some embodiments, themethod comprises the steps of: a) subjecting said culture to a growthperiod comprising exposing said culture to growth conditions to causesaid culture to reproduce, (b) subjecting said culture to apolymerization period comprising exposing said culture to polymerizationconditions to cause said culture to produce intracellular PHA, and (c)repeating step (a) and then second step (b) two or more times.

In some embodiments the growth period comprises a period in which theculture reproduces or otherwise produces biomass and/or reproduces. Insome embodiments, the polymerization period comprises a period in whichthe culture synthesizes PHA. In some embodiments, the growth period andthe polymerization period are induced by the culture media (e.g., theextracellular media around the culture). In some embodiments, alterationin the media conditions induce a transition (partial or complete)between growth and polymerization periods. In some embodiments, aculture is cycled between growth and polymerization periods two, three,four, or more times, in order to produce PHA and then reproduce biomass,which is subsequently used to generate additional PHA.

In some embodiments, the culture is exposed to non-sterile conditions.In certain such embodiments, input carbon is non-sterile. However, insome embodiments, sterile conditions exist. In some embodiments, theculture is dynamic over time in that it may be exposed to extraneousmicroorganisms. In certain embodiments, this is due to a non-sterileculture environment. In some embodiments, extraneous microorganismspotentiate the production of PHA.

In several embodiments, a process for the reduction of pigmentation in amicroorganism culture is provided. In one embodiment, the processcomprises the steps of: providing a medium comprising a microorganismculture comprising dissolved oxygen, and increasing the concentration ofdissolved oxygen over successive periods to select for light-colored orlow-level pigmentation microorganisms.

PHA, while able to be treated post-production to reduce pigmentation, isless expensive to produce when lower levels of pigmentation exist. Somemicroorganisms are more pigmented than others, and therefore in severalembodiments, selection against the more pigmented microorganisms resultsin a less pigmented PHA, which reduces production costs. In someembodiments. the microorganisms cultured and selected for aremethanotrophic microorganisms. By manipulating the culture conditions,which benefit certain varieties of microorganisms, a less pigmentedculture (and hence a less pigmented PHA) result. In some embodiments,increases in concentration of dissolved oxygen over periods ranging from1-3 hours, 3-5 hours, 5-7 hours, 7-10 hours, 10-15 hours, or 15-24 hoursare used to select for less pigmented microorganisms.

In several embodiments, a process for the conversion of a gas into apolyhydroxyalkanoate (PHA) is provided, wherein the process comprisesthe steps of a) providing i) a first gas and ii) a culture ofmicroorganisms, b) contacting the first gas with the culture to causethe culture to convert the first gas into a second gas c) contacting thesecond gas with the culture, and d) causing the culture to use thesecond gas to produce PHA.

In some embodiments, the microorganisms are autotrophic, methanogenic,heterotrophic, or combinations thereof.

In one embodiment the first gas is carbon dioxide. In one embodiment thefirst gas is oxygen. In one embodiment the first gas is methane. In oneembodiment the first gas is a volatile organic compound.

In one embodiment the second gas is carbon dioxide. In one embodimentthe second gas is oxygen. In one embodiment the second gas is methane.In one embodiment the second gas is a volatile organic compound.

It shall be appreciated that the selection of the first and the secondgas is based on the type of microorganism or microorganisms beingcultured.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a block flow diagram comprising the steps of: microorganismfermentation and PHA synthesis, PHA-containing biomass removal,PHA-reduced biomass and isolated PHA production, PHA-reduced biomassrecycling and fermentation, and isolated PHA purification.

DETAILED DESCRIPTION

While PHAs have significant environmental advantages compared to fossilfuel-based plastics, the cost of PHA production is generally viewed as asignificant limitation to the industrial production and commercialadoption of PHAs. Generally, the overall cost of PHA production isdetermined by three major inputs: 1) carbon, 2) chemicals, and 3)energy. Accordingly, efforts to reduce the cost of PHA production mustaddress one or more of these areas, specifically by: i) reducing carboninput costs, ii) increasing carbon-to-PHA yields, iii) reducing thevolume of chemicals required for PHA production, and/or iv) increasingenergy-to-PHA yields.

As discussed above, food crop derived sugars in genetically engineeredmicroorganism-based aqueous fermentation systems are widely regarded asthe most carbon, chemical, energy, and, thus, cost efficient PHAproduction method. Despite these efficiencies, sugar-based PHAproduction remains many times more expensive than fossil fuel-basedplastics production. Attempts to reduce the carbon input cost of the PHAproduction process, by utilizing carbon-containing industrial off-gases,such as carbon dioxide and methane, have been previously limited bytechnical challenges and stoichiometric limitations that render thegas-to-PHA production process significantly more energy and chemicalintensive, and thus more costly, than the food crop-based PHA productionprocess.

Specifically, these technical challenges and stoichiometric limitationsinclude: low mass transfer rates, low microorganism growth rates,extended polymerization times, low cell densities, high oxygen demand(relative to solid substrates), and low PHA cellular inclusionconcentrations. Whereas sugar-based fermentation systems have theability to generate high cellular densities and PHA inclusionconcentrations, carbon-containing gas-based fermentation processestypically cannot, based on fundamental cell morphology and mass transferconstraints, generate cellular and PHA densities exceeding 10-30% ofdensities possible in sugar-based processes. As a result, the ratio ofenergy-to-PHA required to carry out upstream carbon injection, oxygeninjection, system cooling, and culture mixing, as well as downstream PHApurification, significantly exceeds the energy-to-PHA ratio required forsugar-based PHA production methods, thereby rendering theemissions-based process uncompetitive when compared to bothpetroleum-based plastics and sugar-based As.

Several embodiments of the present invention therefore relate to a novelmethod for the production of PHA using carbon-containing gases as asource of carbon, wherein the energy input-to-PHA production ratio,carbon input-to-PHA production ratio, and cost efficiency of the processis significantly improved over previous gas-based PHA productionprocesses.

In several embodiments, this process may be accomplished by a) culturinga first microorganism culture capable of metabolizing the carbon withinboth a carbon-containing gas and biomass, or a derivative thereof, b)manipulating the conditions of the culture to cause the culture toproduce PHA-containing biomass, c) removing a portion of thePHA-containing biomass; d) extracting at least a portion of the PHAwithin the removed PHA-containing biomass to create substantiallyisolated PHA and substantially PHA-reduced biomass, e) purifying theisolated PHA, and f) returning the PHA-reduced biomass to themicroorganism culture to cause the microorganism culture to metabolizethe carbon within the PHA-reduced biomass into PHA.

According to some embodiments, the steps of this process are as follows:(a) providing a microorganism culture comprising biomass and PHA; (b)removing a portion of the PHA-containing biomass from the culture, andextracting PHA from the removed PHA-containing biomass to produceisolated PHA and PHA-reduced biomass; (c) purifying the isolated PHA,and (d) returning the PHA-reduced biomass to be mixed with the cultureto cause the culture to convert the carbon within the PHA-reducedbiomass into PHA. Each of the above recited steps in the process arediscussed in more detail below.

Providing a Microorganism Culture Comprising Biomass and PHA

The terms “microorganism”, “microorganisms”, “culture”, “cultures”, and“microorganism cultures”, as used herein, shall be given their ordinarymeaning and shall include, but not be limited to, a single microorganismand/or consortium of microorganisms, including, among others,genetically-engineered bacteria, fungi, algae, and/or yeast. In someembodiments, microorganisms are naturally occurring and in someembodiments microorganisms are genetically-engineered. In someembodiments, both naturally occurring and genetically-engineeredmicroorganisms are used. In some embodiments, a mixed culture ofmicroorganisms may be used. In some embodiments, microorganisms orcultures shall include a microorganism metabolism system, including theinteractions and/or multiple functions of multiple cultures in one ormore conditions.

The terms “biomass” and “biomass material” shall be given their ordinarymeaning and shall include, but not be limited to, microorganism-derivedmaterial. including intracellular, cellular, and/or extracellularmaterial, such materials including, but not limited to, a polymer orpolymers, amino acids, nucleic acids, carbohydrates, lipids, sugars,PHA, volatile fatty acids, chemicals, gases, such as carbon dioxide,methane, volatile organic acids, and oxygen, and/or metabolicderivatives, intermediaries, and/or end-products. In severalembodiments, biomass is dried or substantially dried.

In some embodiments, the biomass contains less than about 99% water. Inother embodiments, the biomass contains between about 99% to about 75%water, including about 95%, 90%, 85%, and 80%. In some embodiments, thebiomass contains between about 75% and about 25% water, including75%-65%, 65%-55%, 55%-45%, 45%-35%, 35%-25%, and overlapping rangesthereof In additional embodiments, the biomass contains from about 25%water to less than about 0.1% water, including 25%-20%, 20%-15%,15%-10%, 10%-5%, 5%-1%, 1%-0.1%, and overlapping ranges thereof. Instill other embodiments, the biomass contains no detectable amount ofwater. Depending on the embodiment, water is removed from the biomass byone or more of freeze drying, spray drying, fluid bed drying, ribbondrying, flocculation, pressing, filtration, and/or centrifugation. Insome embodiments, the biomass may be mixed with one or more chemicals,such as methylene chloride, acetone, methanol, and/or ethanol, atvarious concentrations. In other embodiments, the biomass may beprocessed through homogenization, heat treatment, pH treatment, enzymetreatment, solvent treatment, spray drying, freeze drying, sonication,or microwave treatment. As used herein, the term “PHA-reduced biomass”shall be given its ordinary meaning and shall mean any biomass whereinat least a portion of PHA has been removed from the biomass. through aPHA extraction process. As used herein, the term “PHA-containingbiomass” shall be given its ordinary meaning and shall mean any biomasswherein at least a portion of the biomass is PHA.

Microorganism cultures useful for the invention described herein includea single strain, and/or a consortium of strains, which are individuallyand/or collectively capable of using carbon containing gases andbiomass, including PHA-reduced biomass, as a source of carbon for theproduction of biomass and PHA. In some embodiments, a microorganismculture according to several embodiments, comprises a microorganismculture that utilizes PHA-reduced biomass, or any derivative thereof,including methanotrophic microorganisms, anaerobic digestion cultures,and other heterotrophic microorganisms, as a source of carbon for theproduction of biomass, or metabolic derivatives including, and inparticular, the production of PHA, protein, methane, and/or carbondioxide (herein, “biomass-utilizing microorganisms”). As used herein,the terms “microorganism”, “culture”, “microorganism culture,”“microorganism system,” “microorganism consortium,” and “consortium ofmicroorganisms” are used interchangeably.

Additionally, any of these terms may refer to one, two, three, or moremicroorganism cultures and/or strains, including a microorganism systemthat is collectively capable of carrying out a complex metabolicfunction (e.g., conversion of PHA-reduced biomass to methane, carbondioxide, protein, and/or PHA). In several embodiments, the microorganismculture comprises of a consortium of carbon-containing gas-utilizingmicroorganisms and a consortium of biomass-utilizing microorganisms. Insome embodiments, the gases metabolized by such cultures comprisemethane, carbon dioxide, and/or a combination thereof.

In some embodiments, the microorganism culture comprises a consortium ofacidogenic, acetogenic, methanogenic, methanotrophic, and/or autotrophicmicroorganisms in one or more individual bioreactors. As such, in someembodiments, the cultures are grown in one or more distinct cultureconditions. In some embodiments, the conditions are either aerobic oranaerobic conditions. In some embodiments, culture conditions are variedover time (e.g. initially aerobic with a transition to anaerobic, orvice versa). As used herein, the term “bioreactor” shall be given itsordinary meaning and shall also refer to a tank, vessel, or anycontainer or device suitable for growth and culturing of microorganisms.

In some embodiments, the microorganism culture is contained within asingle vessel, wherein the steps of converting PHA-reduced biomass tobiomass, converting biomass to PHA, and converting carbon-containinggases to biomass and/or PHA occur simultaneously or sequentially.

In other embodiments, the microorganism culture is contained withinmultiple vessels, which are designed to carry out specific and uniquefunctions. For example, one embodiment includes the steps of (a)converting PHA-reduced biomass to PHA-reduced biomass-derived materialssuch volatile organic acids, methane, and/or carbon dioxide, which iscarried out in a first vessel and (b) synthesizing PHA from PHA-reducedbiomass-derived materials which is carried out in a second, separatetank under independent conditions. In some embodiments, one or more ofthe tanks is an anaerobic digestion tank and one or more other tank isan aerobic fermentation tank.

As used herein, the term “gas-utilizing microorganisms” shall be givenits ordinary meaning and shall refer to microorganisms capable ofutilizing gases containing carbon for the production of biomass,including the production of PHA. Similarly, the terms “methanotrophicmicroorganisms” and “methane-utilizing microorganisms” shall be giventheir ordinary meanings and shall refer to microorganisms capable ofutilizing methane as a source of carbon for the production of biomass.Further, the terms “Autotrophic microorganisms” and “carbondioxide-utilizing microorganisms” shall be given their ordinary meaningand shall refer to microorganisms capable of utilizing carbon dioxide asa source of carbon for the production of biomass, includingmicroorganisms that utilize natural and/or synthetic sources of light tocarry out the metabolism of carbon dioxide into biomass. The term“heterotrophic microorganisms”, as used herein, shall be given itsordinary meaning and shall include methanotrophic, methanogenic,acidogenic, acetogenic and biomass-utilizing microorganisms, includingmicroorganisms that convert sugar, volatile fatty acids, or other carbonsubstrates to biomass. The term “methanogenic microorganisms” shall begiven its ordinary meaning and shall refer to microorganisms thatconvert biomass to methane, including the consortium of microorganismsrequired to carry out such a process, including, but not limited to,acidogenic and acetogenic microorganisms.

As discussed herein, in several embodiments, carbon-containing gases areused as a source of carbon by microorganism cultures. In someembodiments, other sources of carbon are used (e.g., PHA-reducedbiomass), either alone or in combination with carbon-containing gases.In some embodiments, the carbon-containing gases used include, but arenot limited to, carbon dioxide, methane, ethane, butane, propane,benzene, xylene, acetone, methylene chloride, chloroform, volatileorganic compounds, hydrocarbons, and/or combinations thereof. The sourceof the carbon-containing gases depends on the embodiment. For example,carbon-containing gas sources used in some embodiments includelandfills, wastewater treatment plants, anaerobic metabolism, powerproduction facilities or equipment, agricultural digesters, oilrefineries, natural gas refineries, cement production facilities, and/oranaerobic organic material digesters, including both solid and liquidmaterial digesters.

In several embodiments described herein, microorganisms may include, butare not limited to, yeast, fungi, algae, and bacteria (includingcombinations thereof). Suitable yeasts include, but are not limited to,species from the genera Candida, Hansenula, Torulopsis, Saccharomyces,Pichia, 1-Debaryomyces, Lipomyces, Cryptococcus, Nernatospora, andBrettanomyces. Suitable genera include Candida, Hansenula, Torulopsis,Pichia, and Saccharomyces. Examples of suitable species include, but arenot limited to: Candida boidinii, Candida mycoderma, Candida utilis,Candida stellatoidea, Candida robusta, Candida claussenii, Candidarugosa, Brettanomyces petrophilium, Hansenula minuta, Hansenula satumus,Hansenula californica, Hansenula mrakii, Hansenula silvicola, Hansenulapolymorpha, Hansenula wickerhamii, Hansenula capsulata, Hansenulaglucozyma, Hansenula henricii, Hansenula nonfermentans, Hansenulaphilodendra, Torulopsis candida, Torulopsis bolmii, Torulopsisversatilis, Torulopsis glabrata, Torulopsis molishiana, Torulopsisnemodendra, Torulopsis nitratophila, Torulopsis pinus, Pichia farinosa,Pichia polymorpha, Pichia membranaefaciens, Pichia pinus, Pichiapastoris, Pichia trehalophila, Saccharomyces cerevisiae, Saccharomycesfragilis, Saccharomyces rosei, Saccharomyces acidifaciens, Saccharomyceselegans, Saccharomyces rouxii, Saccharomyces lactis, and/orSaccharomyces fractum.

Suitable bacteria include, but are not limited to, species from thegenera Bacillus, Mycobacterium, Actinomyces, Nocardia, Pseudomonas,Methanomonas, Protaminobacter, Methylococcus, Arthrobacter,Methylomonas, Brevibacterium, Acetobacter, Methylomonas, Brevibacterium,Acetobacter, Micrococcus, Rhodopseudomonas, Corynebacterium,Rhodopseudomonas, Microbacterium, Achromobacter, Methylobacter,Methylosinus, and Methylocystis. Preferred genera include Bacillus,Pseudomonas, Protaminobacter, Micrococcus, Arthrobacter and/orCorynebacterium. Examples of suitable species include, but are notlimited to: Bacillus subtilus, Bacillus cereus, Bacillus aureus,Bacillus acidi, Bacillus urici, Bacillus coagulans, Bacillus mycoides,Bacillus circulans, Bacillus megaterium, Bacillus licheniformis,Pseudomonas ligustri, Pseudomonas orvilla, Pseudomonas methanica,Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas oleovorans,Pseudomonas putida, Pseudomonas boreopolis, Pseudomonas pyocyanea,Pseudomonas methylphilus, Pseudomonas brevis, Pseudomonas acidovorans,Pseudomonas methanoloxidans, Pseudomonas aerogenes, Protaminobacterruber, Corynebacterium simplex, Corynebacterium hydrocarbooxydans,Corynebacterium alkanum, Corynebacterium oleophilus, Corynebacteriumhydrocarboclastus, Corynebacterium glutamicum, Corynebacterium viscosus,Corynebacterium dioxydans, Corynebacterium alkanum, Micrococcuscerificans, Micrococcus rhodius, Arthrobacter rufescens, Arthrobacterparafficum, Arthrobacter citreus, Methanomonas methanica, Methanomonasmethanooxidans, Methylomonas agile, Methylomonas albus, Methylomonasrubrum, Methylomonas methanolica, Mycobacterium rhodochrous,Mycobacterium phlei, Mycobacterium brevicale, Nocardia salmonicolor,Nocardia minimus, Nocardia corallina, Nocardia butanica,Rhodopseudomonas capsulatus, Microbacterium ammoniaphilum,Archromobacter coagulans, Brevibacterium butanicum, Brevibacteriumroseum, Brevibacterium flavum, Brevibacterium lactoferrnentum,Brevibacterium paraffinolyticum, Brevibacterium ketoglutamicum, and/orBrevibacterium insectiphilium.

In several embodiments, more than one type or species of microorganismis used. For example, in some embodiments, both algae and bacteria areused. In some embodiments, several species of yeast, algae, fungi,and/or bacteria are used. In some embodiments, a single yeast, algae,fungi, and/or bacteria species is used. In some embodiments, aconsortium of cyanobacteria is used. In some embodiments, a consortiumof methanotrophic microorganisms is used. In still additionalembodiments, a consortium of both methanotrophic bacteria andcyanobacteria are used. In several embodiments, methanotrophic,heterotrophic, methanogenic, and/or autotrophic microorganisms are used.

In several embodiments of the invention, the microorganism culturecomprises a consortium of methanotrophic, autotrophic, and/orheterotrophic microorganisms, wherein methane and/or carbon dioxide isindividually, interchangeably, or simultaneously utilized for theproduction of biomass. In some embodiments, PHA-reduced biomass is usedas a source of carbon by heterotrophic, autotrophic, and/ormethanotrophic microorganisms. In several embodiments of the invention,the microorganism culture comprises methanotrophic microorganisms,cyanobacteria, and non-methanotrophic heterotrophic microorganisms,wherein methane and carbon dioxide are continuously utilized as sourcesof carbon for the production of biomass and PHA.

In some embodiments, microorganisms are employed in a non-sterile, open,and/or mixed environment. In other embodiments, microorganisms areemployed in a sterile and/or controlled environment.

The terms “PHA”, “PHAs”, and “polyhydroxyalkanoate”, as used herein,shall be given their ordinary meaning and shall include, but not belimited to, polymers generated by microorganisms as energy and/or carbonstorage vehicles; biodegradable and biocompatible polymers that can beused as alternatives to petrochemical-based plastics such aspolypropylene, polyethylene, and polystyrene; polymers produced bybacterial fermentation of sugars, lipids, or gases; and/or thermoplasticor elastomeric materials derived from microorganisms. PHAs include, butare not limited to, polyhydroxybutyrate (PHB), polyhydroxyvalerate(PHV), pol yhydroxybutyrate-coval crate (PHB/V), andpolyhydroxyhexanoate (PHHx), as well as both short chain length (SCL),medium chain length (MCL), and long chain length (LCL) PHAs.

The terms “growth-culture medium”, “growth medium”, “growth-culturemedia”, “medium”, and “media”, as used herein shall be given theirordinary meaning and shall also refer to materials affecting the growth,metabolism, PHA synthesis, and/or reproductive activities ofmicroorganisms. Non-limiting examples of growth-culture media used inseveral embodiments include a mineral salts medium, which may comprisewater, nitrogen, vitamins, iron, phosphorus, magnesium, and variousother nutrients suitable to effect, support, alter, modify, control,constrain, and/or otherwise influence the metabolism and metabolicorientation of microorganisms. A growth-culture medium may comprisewater filled with a range of mineral salts. For example, each liter of aliquid growth-culture medium may be comprised of about 0.7-1.5 g KH₂PO₄,0.7-1.5 g K₂HPO₄, 0.7-1.5 g KNO₃, 0.7-1.5 g NaCl, 0.1-0.3 g MgSO₄, 24-28mg CaCl₂*2H₂O, 5.0-5.4 mg EDTA Na₄(H₂O)₂, 1.3-1.7 mg FeCl₂*4H₂O,0.10-0.14 mg CoCl₂*6H₂O, 0.08-1.12 mg MnCl₂*2H₂O, 0.06-0.08 mg ZnCl₂,0.05-0.07 mg H₃BO₃, 0.023-0.027 mg NiCl₂*6H₂O, 0.023-0.027 mgNaMoO₄*2H₂O, and 0.011-0.019 mg CuCl₂*2H₂O. A growth-culture medium canbe of any form, including a liquid, semi-liquid, gelatinous, gaseous,foam, or solid substrate.

In several embodiments of the invention, a microorganism culture isproduced in a liquid growth medium, wherein carbon dioxide and methaneare utilized as a gaseous source of carbon for the production ofmethanotrophic and/or autotrophic biomass. In some embodiments,PHA-reduced methanotrophic and/or PHA-reduced autotrophic biomass isutilized as a source of carbon for the production of heterotrophicbiomass and heterotrophically-produced PHA. In some embodiments, thegrowth medium is manipulated to effect the growth, reproduction, and PHAsynthesis of the microorganism culture. Methods for the production ofmethanotrophic microorganisms are disclosed in the art, and aredescribed by Herrema, et al., in U.S. Pat. No. 7,579,176, which ishereby incorporated by reference in its entirety. Methods for theproduction of cyanobacteria are described by Lee, et al. (“High-densityalgal photobioreactors using light-emitting diodes,” Biotechnology andBioengineering, Vol. 44, Issue 10, pp. 1161-1167), which is herebyincorporated by reference in its entirety. Methods for the production ofmethane from biomass are described by Deublein, et al. (“Biogas fromWaste and Renewable Resources, WILEY-VCH Verlag GmbH & Co. KgaA, 2008),which is hereby incorporated by reference in its entirety. In someembodiments, PHA synthesis may be effected through the manipulation ofone of more elements of the culture medium, including through thereduction, increase, or relative change in either the total orbioavailable concentration of one or more of the following elements:nitrogen, phosphorus, oxygen, methane, carbon dioxide, magnesium,potassium, iron, copper, sulfate, manganese, calcium, chlorine, boron,zinc, aluminum, nickel, and/or sodium. Methods for the production of PHAare described by Herrema, et al., in U.S. Pat. No. 7,579,176.

In several embodiments of the invention, methanol is added to a cultureof methanotrophic microorganisms utilizing a closed loop recycling gasstream comprising methane. In some embodiments, methanotrophicmicroorganisms are enabled to grow under conditions of, and consume,very low concentrations of methane by co-utilizing methanol as a carbonsubstrate. In the past, the growth of methanotrophic microorganisms wassignificantly reduced under low methane concentrations due to, amongother things, low mass transfer rates. In some embodiments, by theaddition of methanol in a closed loop gas recycling system, it ispossible to effect the substantially complete elimination of methane bymethanotrophic microorganisms.

In several embodiments of the invention, the diffusion of light isincreased in a liquid growth culture media by reducing the density ofthe liquid in a light path. In some embodiments the culture comprisesautotrophic microorganisms. In some embodiments, the application of gasbubbles into the media decreases the relative solids density of thelight path, thus enabling an increased diffusion of light into a liquidculture media from a given light intensity energy.

In several embodiments of the invention, a series of submerged lightrods are placed into a liquid culture to manipulate or adjust theculture conditions. In some embodiments, the culture comprisesautotrophic microorganisms. In some embodiments, the light rods functionto diffuse light, diffuse gas, act as static or dynamic mixers, assistin the circulation of a liquid culture media, and/or facilitate heatexchange through the circulation of a gas, liquid, and/or combinationthereof.

Traditionally, pH control in a microorganism growth system is difficultand/or costly to maintain. In some embodiments, pH is controlled byvarying the nitrogen source supplied to a microorganism growth systembetween pH-increasing and pH-reducing nitrogen sources, e.g., NO₃ ⁻ andNH₃ ⁺, respectively. In some embodiments, nitrogen sources are utilizedthat do not significantly affect the pH of the system, including, whenapplicable, complex nitrogen sources such as biomass. In additionalembodiments, a closed loop system is employed to reduce changes in pH.In some embodiments, respiration-generated carbon dioxidecounterbalances increases in pH caused by the utilization ofpH-increasing nitrogen sources, such as nitrates.

A number of methods are known for the induction of gas into liquid,including static mixing, ejector mixing, propeller mixing, and/or acombination thereof. Simultaneously, it is also known that shear can behighly detrimental to microorganism growth, and can often impede orpermanently deactivate microorganism metabolism. Thus, mass transfer ina gas-based system is often limited by the need to counterbalancesufficient mixing with shear considerations. In several embodiments ofthe invention, a vessel comprising liquid culture media is mixed with agas, e.g. methane, under relatively high shear conditions, and thensubsequently transferred to a vessel comprising liquid culture mediamaintained under relatively low shear conditions. In some embodiments,microorganism growth is primarily induced in the low shear vessel. Insome embodiments, high gas transfer rates are effected in the first highshear vessel by mixing while performed in the low shear vessel bygaseous diffusion.

In another embodiment, a closed loop gas recycling system is maintained,wherein a vessel comprising gas-utilizing microorganisms is suppliedwith gas, wherein the gas is utilized by gas-utilizing microorganisms,and wherein the rate at which gas is added to the system is determinedby the rate at which the pressure in the vessel changes in accordancewith the conversion of gases into metabolic derivatives (such asbiomass, carbon dioxide, and water). For example, a vessel containingmethane-utilizing microorganisms may be pressurized to 60 psi with acombination of methane and oxygen; as the pressure in the system dropsin accordance with the metabolism of the methane-utilizingmicroorganisms, additional methane and oxygen is added to the systemsuch that the pressure of the vessel remains at 60 psi. It shall beappreciated that, in certain embodiments, higher or lower pressures aremaintained. In some embodiments, the system is periodically flushed toremove carbon dioxide. In some embodiments, autotrophic microorganismsand a light injection system may be added to the system in order toconvert carbon dioxide into additional oxygen, thereby substantiallyreducing or eliminating the need to flush the system and/or introduceoxygen.

In several embodiments, PHA synthesis is induced in a microorganismculture comprising methane-utilizing, heterotrophic, and/or carbondioxide-utilizing microorganisms wherein a PHA inclusion concentration(by dry biomass weight) is generated of between about 0.01% and 95%. Insome embodiments, the inclusion concentration is between 25% and 80%,including 25-35%, 35% to 50%, 50% to 65%, 65% to 80%, and overlappingranges thereof. In some embodiments, the inclusion concentration isbetween 0.01% and 55%, including, 0.01% to 1%, 1% to 5%, 5% to 10%, 10%to 15%, 15% to 20%, 20%, to 25%, 25% to 30%, 30% to 35%, 35% to 40%, 40%to 45%, 45% to 50%, 50% to 55%, and overlapping ranges thereof. In someembodiments, PHA synthesis is induced in a methanotrophic,heterotrophic, and/or autotrophic microorganism culture wherein a PHAinclusion concentration (by dry biomass weight) is generated of between20% and 80%, between 30% and 70%, between 40% and 60%, between 50% and70%, including 50% to 55%, 55 to 60%, 60% to 65%, 65% to 70%, andoverlapping ranges thereof. In some embodiments of the invention, PHAsynthesis is induced in microorganism culture comprising methanotrophic,autotrophic, and heterotrophic microorganisms, wherein an average PHAinclusion concentration (by dry biomass weight) is greater than 5%,greater than 20%, greater than 40%, greater than 65%, or greater than70% by dry cell weight.

In some embodiments, the growth culture media is manipulated to induceboth i) microorganism growth and ii) PHA synthesis within one or moreopen, non-sterile, or sterile vessels using a feast-famine cultureregime. In some embodiments, the microorganisms are subject tosuccessive alternating periods of nutrient/carbon availability andnutrient/carbon unavailability to encourage the reproductive success ofmicroorganisms that are capable of synthesizing PHA, particularly athigh inclusion concentrations. Feast-famine regimes useful for theselection of PHA producing microorganisms, including PHA-producingmethanotrophic microorganisms, over microorganisms that either cannotproduce PHA, produce PHA slowly, or produce PHA at relatively lowconcentrations are described in the art (Frigon, et al., “rRNA andPoly-Hydroxybutyrate Dynamics in Bioreactors Subjected to Feast andFamine Cycles,” Applied and Environmental Microbiology, April 2006, p.2322-2330; Muller, et al., “Adaptive responses of Ralstonia eutropha tofeast and famine conditions analysed by flow cytometry,” J Biotechnol.1999 October 8;75(2-3):81-97; Reis, et al., “Production ofpolyhydroxyalkanoates by mixed microbial cultures,” Bioprocess andBiosystems Engineering, Volume 25, Number 6, 377-385, DOI:10.1007/s00449-003-0322-4.)

In some embodiments of the invention, the classical feast-famine regimeis modified to reduce PHA losses. Specifically, in the past,feast-famine regimes were thought to be effective by passing amicroorganism culture through a period wherein carbon or nutrients wereunavailable or relatively limited for metabolism, thereby forcing theculture to accumulate and/or consume intracellular PHA as a source ofcarbon to survive, and thereby selecting for microorganisms with thecapacity to synthesize and store PHA. Applicant has surprisinglydiscovered that, some microorganisms with higher concentrations ofintracellular PHA reproduce more efficiently than microorganisms withlower concentrations of intracellular PHA in periods of carbonavailability and nutrient balance. Thus, in one embodiment, a novel PHAproduction regime is employed in one or more vessel whereinmicroorganisms are subjected to two successive and recurring phases: 1)growth, wherein carbon and nutrient availability is optimized forreproduction, and 2) PHA synthesis, wherein carbon is available inexcess, and one or more nutrient is reduced or increased relative to thegrowth period to induce PHA synthesis. In some embodiments, a fractionof the vessel media is removed for downstream PHA extraction andprocessing after the PHA synthesis period, and that fraction is replacedwith lower cell density media, which simultaneously returns carbon andnutrient concentrations to reproductively favorable levels, e.g., thegrowth phase or growth conditions, and causes microorganisms to enterinto a reproductive phase without consuming significant portions ofintracellular PHA. As such, efficient PHA producing microorganismsselectively reproduce over inefficient or non-PHA producingmicroorganisms. As a result, some embodiments, i) increase the speed ofthe microorganism selection process by removing the PHA consumption steptypical to previous feast famine models and ii) reduce the loss of PHAto cellular metabolism. According to such embodiments, the feast faminemodel is converted to a feast-polymerization-feast process. In severalembodiments methanotrophic, autotrophic, and/or heterotrophic culturesare used in the feast-polymerization-feast process.

Removing A Portion Of The PHA-Containing Biomass From The Culture, AndExtracting PHA From The Removed PHA-Containing Biomass To ProduceIsolated PHA And PHA-Reduced Biomass

In several embodiments, following the production of a microorganismculture comprising biomass and PHA (discussed above), at least a portionof the PHA-containing biomass is removed from the culture. In severalembodiments, a portion ranging from 20% to 80% of the PHA-containingbiomass is removed, including 30%-70%, 40% to 60%, 45% to 55%, andoverlapping ranges thereof. Removal of PHA-containing biomass may beperformed by a number of methods, including centrifugation, filtration,density separation, flocculation, agglomeration, spray drying, or otherseparation technique. In some embodiments, dewatering (e.g., bycentrifugation) results in a biomass having a desirable water contentthat facilitates downstream processing of the biomass. For example, insome embodiments, centrifugation of the PHA-containing biomass reducesthe amount of culture media (increases the relative biomassconcentration) to a concentration range between about 100 and 500 gramsof biomass per liter of culture media. In some embodiments, theconcentration is of the biomass is adjusted to about 100 to 200 g/L, 200to 300 g/L, 300 to 400 g/L, 400 to 500 g/L, and overlapping rangesthereof. Advantageously, such an approach also produces, as an effectiveby-product, clarified culture media that can be optionally treated,measured, or recycled into one or more culture vessels.

In some embodiments, after a portion of the PHA-containing biomass isremoved from the culture, PHA is extracted from the removedPHA-containing biomass to produce isolated PHA and PHA-reduced biomass.

As used herein, the terms “extraction” and “PHA extraction” shall begiven their ordinary meaning and shall be used interchangeably todescribe the removal and/or separation of PHA from biomass. PHAs may beextracted from biomass by several processes, including, but not limitedto, the use of chemicals, mechanical means, solvents, and enzymes. Theseprocesses include the use of: i) solvents, such as acetone, ethanol,methanol, methylene chloride, and dichloroethane, with and/or withoutthe application of pressure and/or elevated temperatures, ii)supercritical carbon dioxide, iii) enzymes, such as protease, iv)surfactants, v) pH adjustment, including the protonic or hydroxide-baseddissolution of non-PHA biomass, and/or vi) hypochlorite to dissolvenon-PHA biomass, including the use of hypochlorite in conjunction with asolvent, such as methylene chloride. In some embodiments of theinvention, PHA is extracted by solvent extraction from a PHA-containingbiomass comprising gas-utilizing microorganisms and/or biomass-utilizingmicroorganisms to produce isolated PHA and PHA-reduced biomass. In somesolvent-based embodiments, solvents suitable for dissolving the PHA areused, including carbon dioxide, acetone, methylene chloride, chloroform,water, ethanol, and methanol. In some embodiments, particular ratios ofsolvent to PHA provide optimal dissolution of PHA from the culture, andtherefore lead to improved extraction and isolation efficiency andyield. For example, in some embodiments, ratios of solvent to PHA (ingrams) of about 500:1 are used. In some embodiments, ratios of about0.01:1 are used. In some embodiments, ratios ranging from between about500:1 and 0.01:1 are used, such as 0.05:1, 1.0:1, 1.5:1, 20:1, 250:1,300:1, 350:1, 400:1, or 450:1.

As discussed above, changes in temperature and/or pressure may also beused to facilitate the extraction of PHA from the PHA-containingbiomass. In some embodiments, the extraction solvent chosen dictates thelimits of temperature, pressure, and/or incubation times that are used.In some embodiments, solvent is combined with PHA-containing biomass andincubated for several minutes up to several hours. For example, in someembodiments, incubation is for about 10 minutes, while in otherembodiments, overnight incubation times are used. In some embodiments,incubation times range from 30 minutes to about 1 hour, about 1 hour toabout 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6hours, about 6 hours to about 8 hours, about 8 hours to about 10 hours,and from about 10 hours to overnight. Choice of incubation time isdetermined by solvent, culture density (e.g., number of microorganisms),type of organisms, expected PHA yield, and other similar factors.

Incubation temperature is also tailored to the characteristics of agiven culture. Incubation temperatures can range from below roomtemperature to elevated temperatures of up to about 150° C. or about200° C. For example, depending the solvent and other variables of theculture, temperatures are used that range from about 10° C. to 25° C.,from about 25° C. to about 40° C. , from about 40° C. to about 55° C. ,from about 55° C. to about 60° C., from about 60° C. to about 75° C.,from about 75° C. to about 90° C., from about 90° C. to about 105° C.,from about 105° C. to about 120° C., from about 120° C. to about 135°C., from about 135° C. to about 150° C., from about 150° C. to about200° C., and overlapping ranges thereof.

As can be appreciated, if changes in temperature are made to a culturein a closed vessel, changes in pressure result. In some embodiments,increased pressure provides a shearing effect that aids in theliberation of PHA from the microorganisms. In some embodiments, pressureis regulated within a particular range. For example, in someembodiments, pressure of the reaction of the PHA-containing biomass withsolvent occurs between about 40 and 200 psi, including about 50 to 60psi, 60 to 70 psi, 70 to 80 psi, 80 to 90 psi, 90 to 100 psi, 100 to 125psi, 125 to 150 psi, 150 to 175 psi, 175 to 200 psi and overlappingranges thereof. Additional sources of shear (e.g., agitation, pumping,stirring etc.) are optionally used in some embodiments to enhance theextraction of PHA. Any one, or combination, of the PHA extractionmethods described herein, or disclosed in the art, may be utilized as amethod to carry out PHA extraction and remove PHA from thePHA-containing biomass.

In several embodiments, a solvent-based extraction system is utilized tocarry out PHA extraction. In some embodiments, solvents are utilized tocarry out PHA extraction at high temperatures, wherein PHA extractionoccurs simultaneous with a temperature-enhanced breakdown or dissolutionof PHA-containing biomass. In some embodiments, one or more solvent isutilized that is biodegradable and metabolically assimilable by theculture, such that PHA-reduced biomass comprising biomass and one ormore biodegradable solvent may be contacted with the culture, and boththe PHA-reduced biomass and the solvent may be utilized by the cultureas a source of carbon. Non-limiting examples of such solvents includecarbon dioxide, acetone, ethanol, methanol, and methylene chloride,among others.

In several embodiments a mixture of solvent and PHA comprises multiplephases, e.g. an aqueous phase and an organic phase. In some embodiments,solvent-based extraction comprises a more uniform mixture of solvent andPHA. In some embodiments, depending on the solvent the phases areseparated prior to recovery of the PHA. In some embodiments,centrifugation is employed to further distinguish and separate thephases of the mixture (e.g., separation of the solvent-PHA phase fromthe water-biomass phase). In some embodiments, heat is also employed tomaintain the solubility of the PHA in a given solvent.

It shall be appreciated that the solubility of PHA varies with thesolvent used, and therefore the temperature (if adjusted) and theseparation techniques are tailored to match the characteristics of agiven solvent. Thus, in some embodiments employing centrifugation, forexample, a low speed centrifugation is used to separate the solvent-PHAphase from the water-biomass phase. In other embodiments, depending onthe solvent, higher speed centrifugation is used. In some embodiments,centrifugation is employed in stages, e.g., low speed centrifugationfollowed by high speed centrifugation. Any of a variety of centrifugescan be employed, depending on the solvent used, for example, basketcentrifuges, swinging bucket centrifuges, fixed rotor centrifuges,disc-back centrifuges, supercentrifuges, or ultracentrifuges.

In some embodiments, adjustable discharge ports suitable for aparticular centrifuge are used in order to control the rate and degreeof separation of solvent-PHA phase from the water-biomass phase. In someembodiments, the concentration of water in the water-biomass phase isadjusted to allow for suitable flow of the mixture through thecentrifuge (or within a centrifuge tube). For example, in someembodiments, flow is suitable for separating the phases when theconcentration of biomass (relative to water) is between about 1 and 100g/L. In some embodiments, the concentration is between about 10 to 20g/L, 20 to 30 g/L, 30 to 40 g/L, 40 to 50 g/L, 50 to 60 g/L, 60 to 70g/L, 70 to 80 g/L, 80 to 90 g/L, 90 to 100 g/L, 100 to 200 g/L, 200 to400 g/L, 400 to 600 g/L, and overlapping ranges thereof.

In still additional embodiments, increases in temperature not onlyfacilitate the extraction of the PHA, they also facilitate the isolationof the PHA from the solvent (e.g. increased temperature increasessolvent evaporation).

In some embodiments, an extraction process is carried out to remove PHAfrom a microorganism in such a manner that the microorganism isdeactivated. In some embodiments, the deactivation is permanent, whilein some embodiments the deactivation is temporary. Without being boundby theory, it is believed that PHA extraction techniques which do notpermanently disable microorganisms enable the PHA-reduced biomassgenerated thereby to contribute to the metabolism of carbon sourcesafter a PHA extraction process, including through intracellular andextracellular metabolism. In one embodiment, methods useful for thetemporary disablement of microorganisms include solvent extraction,including solvent extraction carried out below 100 ° C., andparticularly at intracellular temperatures below 100 ° C., includingextraction temperatures of about 10° C. to 30° C. to 50° C. to 60 ° C.,60 ° C. to 70 ° C., 70 ° C. to 80 ° C., 80 ° C. to 90 ° C., 90 ° C. to100 ° C., and overlapping ranges thereof.

In several embodiments, the PHA concentration of PHA-containing biomassis reduced as a result of the PHA extraction process. In severalembodiments, the PHA concentration of PHA-containing biomass is reducedby at least 0.01% (by dry cell weight). In some embodiments, the PHAconcentration is reduced by about 10%-50%, 50% to 70%, 70% to 75%, 75%to 80%, 80% to 85%, 85%, to 90%, 90% to 95%, 95%-99.9%, and overlappingranges thereof.

While a variety of methods are known to enable the extraction PHA frombiomass, most methods can be categorized into one of two classes: a)solvent-based extraction, or b) NPCM (non-polymer cellular material)dissolution-based extraction. NPCM dissolution-based extraction methodsutilize chemicals (such as hypochlorite, or bleach), enzymes (such asprotease), heat (especially to reach temperatures above 100° C.), pH(acids and bases), and/or mechanical means (such as homogenization) tobreak down, oxidize, and/or emulsify non-PHA cellular material. In somecases, extraction methods from both categories can be combined, such asthe simultaneous utilization of hypochlorite and methylene chloride.

NPCM dissolution-based extraction methods require continuous andnon-recoverable chemical inputs, such as hypochlorite, peroxides,enzymes, and pH adjustors, and also generate significant waste disposalissues. Thus, while these methods are effective, the use ofsolvent-based extraction methods is generally preferred in the industrydue to the capacity of solvents to be distilled and recovered forcontinuous re-utilization in a closed loop cycle. Unfortunately, despiteits benefits, some solvent-based extraction methods are energy intensiveprocesses that play a major role in the high cost of PHA production,often accounting for more than 50% of total production costs.Accordingly, there exists a significant need for a novel method tosignificant increase the energy efficiency of solvent-based extraction.

In several embodiments, a process for the extraction ofpolyhydroxyalkanoates from biomass using a solvent-based extractionmethod is provided, wherein the energy required to carry out the processis reduced relative to prior solvent-based extraction methods.Specifically, in one embodiment a high efficiency PHA extraction processis provided comprising providing a PHA-containing biomass comprising PHAand water, mixing the biomass with a solvent at a temperature sufficientto dissolve at least a portion of the PHA into the solvent and at apressure sufficient to enable substantially all or part of the solventto remain in liquid phase, thereby producing a PHA-lean biomass phaseand a PHA-rich solvent phase comprising solvent, water, and PHA,separating the PHA-rich solvent phase from the PHA-lean biomass phase ata temperature and pressure sufficient to enable substantially all orpart of the solvent to remain in the liquid phase and preventsubstantially all or part of the PHA within the PHA-rich solvent phasefrom precipitating, reducing the pressure or increasing the temperatureof the PHA-rich solvent phase to cause the solvent to vaporize and thePHA to precipitate or become a solid while maintaining the temperatureand/or the pressure of the PHA-rich solvent phase to prevent all or partof the temperature-dependent precipitation of the PHA into water, andcollecting the solid PHA material, including optionally separating theprecipitated PHA from the solvent and/or the water.

In the past, PHA precipitation has been induced in PHA-rich solvent bya) adding a non-PHA solvent to the solvent phase to reduce thesolubility of PHA in the solvent phase and/or b) reducing thetemperature of the solvent phase to reduce the solubility of PHA in thesolvent. In particular, some methods 1) dissolve PHA in a solvent byincreasing the temperature of the solvent and 2) precipitate PHA byreducing the temperature (and, thus, solubility) of the solvent. Othermethods require adding water to a solution of PHA-rich solventcomprising dissolved PHA, wherein the addition of water to the solutionreduces the solubility of the PHA in the solvent and causes the PHA toprecipitate into the solvent and/or water. (For example, U.S. Pat. No.4,562,245; U.S. Pat. No. 4,968,611; U.S. Pat. No. 5,894,062; U.S. Pat.No. 4,101,533, all herein incorporated by reference.) In each of thesecases, energy efficiency is compromised; specifically, by adding wateror a non-PHA solvent to reduce the PHA solubility of a solvent,additional energy is required for downstream water/non-solvent removal,heating, and/or distillation. By reducing the temperature of the solventto reduce the solubility of the solvent and induce PHA precipitation,heat energy is redundantly expended, as the solvent must be re-heatedfor distillation and recovery. Therefore, in several embodiments, ratherthan adding a non-solvent to a PHA-solvent or reducing the temperatureof the PHA-solvent to effect PHA precipitation, pressure and/or anincrease in temperature is used to induce the precipitation orsolidification of the PHA without redundantly reducing the temperatureof solvent. Thus, in such embodiments, there is a significant reductionin the energy required to heat and/or distill non-solvent and/or solventin downstream PHA processing.

The removal of non-PHA materials from PHA often accounts for asignificant fraction of PHA production costs. As a specific example,pigments often cause unwanted discoloration of PHA, and must be removedthrough costly processes, such as ozonation, peroxide washing, acetonewashing, ethanol washing, solvent refluxing, hypochlorite digestion,enzymatic degradation, surfactant dissolution, or other methodsdisclosed in the art. In several embodiments, a non-sterile process isused to select for microorganisms exhibiting minimal pigmentation.Applicant has surprisingly discovered that, by manipulating theconcentration of dissolved oxygen in a microorganism system, a culturemay be selected wherein white, tan, off-white, light brown, and/or lightyellow pigments are exhibited rather than purple, red, pink, dark brown,orange, or other heavy pigments. Specifically, in some embodiments, anexcess of dissolved oxygen is introduced into a growth media oversuccessive periods, resulting in selective vsowth of strains ofmethanotrophic microorganisms which produce white, tan, off-white, lightbrown. and light yellow pigments, rather than those producing pink, red,purple, dark yellow, dark brown, and/or other heavy pigments. As aresult, such embodiments, reduce the need for costly downstreampigmentation removal.

As used herein, the term “PHA-reduced biomass” or “substantiallyPHA-reduced biomass” shall be given its ordinary meaning and shall beused to describe a biomass material wherein the concentration of PHArelative to non-PHA material has been reduced in a PHA-containingbiomass through the utilization of a PHA extraction process. In someembodiments, PHA-reduced biomass is further treated in a variety ofways. In some embodiments, the further treatment includes, but is notlimited to, one or more of dewatering, chemical treatment, sonication,additional PHA extraction, homogenization, heat treatment, pH treatment,hypochlorite treatment, microwave treatment, microbiological treatment,including both aerobic and anaerobic digestion, solvent treatment, waterwashing, solvent washing, and/or drying, including simple or fractionaldistillation, spray drying, freeze drying, rotary drying, and/or ovendrying.

In one embodiment, PHA-reduced biomass is substantially dried, whereinthe resulting dried material comprises less than about 99% liquids,including water, solvents, salts, and/or growth-culture media. In someembodiments, the drying processes disclosed herein yield a driedmaterial containing between about 95% and 75% liquids, between about 75%and 50% liquids, between about 50% and 25% liquids, between about 25%and 15% liquids. between about 15% and 10% liquids, between about 10%and 1% liquids, and overlapping ranges thereof. In some embodiments,drying is complete (e.g., between 1% 0.1% liquids, or less). In anotherembodiment of the invention, a liquid phase comprising PHA-reducedbiomass is subjected to filtration, centrifugation, densitydifferentiation, or other method to increase the solids content of thePHA-reduced biomass.

Traditionally, the separation of biomass from liquid growth media isdifficult and impractical due to the plugging and foulingcharacteristics of biomass. In several embodiments, a method enablingthe efficient filtration of microorganisms is provided. In someembodiments, a liquid chemical is added to the growth media comprisingmicroorganisms. wherein the liquid chemical is ethanol, acetone,methanol. methylene chloride, ketones, alcohols, and/or chlorinatedsolvents, or a combination thereof. In some embodiments, microorganismsare then efficiently separated from liquid growth media using standardfiltration equipment, such as a Buchner filter, filter press, or similarapparatus. In one embodiment, approximately 2 parts acetone are mixedwith one part water, including both intracellular and extracellularwater, to effect the efficient filtration of microorganisms comprisingthe water.

As used herein, the terms “isolated PHA” and “substantially isolatedPHA” shall be given their ordinary meaning and shall refer to PHA thathas been removed from a biomass material as a result of an extractionprocess, or a biomass material wherein the concentration of PHA relativeto non-PHA material has been increased by an extraction process. Inseveral embodiments, isolated PHA is further treated in one or more of avariety of ways, including, but not limited to, purification,filtration, washing, oxidation, odor removal, pigment removal, lipidremoval, non-PHA material removal, and/or drying, includingcentrifugation, filtration, spray drying, freeze drying, simple orfractional distillation, or density differentiation. Methods for thepurification of PHA include the use of peroxides, water, hypochlorite,solvents, ketones, alcohols, and various other agents to separate andremove non-PHA material from PHA material. In some embodiments, PHA isremoved from a microorganism culture by solvent extraction to produceisolated PHA in a PHA-rich solvent phase and PHA-reduced biomass in aPHA-lean liquid phase. In some embodiments, the solvent phase isseparated from the liquid phase by filtration or centrifugation. In someembodiments, both centrifugation and filtration are used in combination(e.g., sequentially). In some embodiments, centrifugation is optionallyfollowed by filtration. In other embodiments, filtration is optionallyfollowed by centrifugation. Filtration, in some embodiments is performedunder vacuum pressure, via gravity feed, under positive pressure, or inspecialized filtration centrifuges. In some embodiments, the filter poresize is adjusted based on the species composition of the microorganismculture. In some embodiments, pore sizes of up to 200 μm are used. Insome embodiments, smaller pore sizes are used, for example 15 to 20 μm,10 to 15 μm, 5 to 10 μm, 1 to 5 μm, 0.001 to 1 μm, and overlappingranges thereof.

In addition to the steps outlined above, additional steps may be takento remove solvent from the extracted PHA, including evaporation, solventcasting, steam stripping, heat treatment, and vacuum treatment, each ofwhich may be preferential, cost-effective, time-effective, oradvantageous depending on the volatility and type of solvent used. Inother embodiments, active processes can be used to reduce the solventcontent of the solvent-PHA mixture. For example, in certain embodiments,alterations in temperature of certain solvents change the solubility ofthe PHA in the solvent, which effectively removes solvent from the PHA(e.g., the solvent is now separable from a precipitated PHA). In someembodiments, filtration, solvent temperatures, or vacuum treatment canbe increased to reduce a portion of the solvent. In some embodiments,solvent to PHA ratios post extraction, filtration, evaporation, solventcasting, steam stripping, heat treatment, and/or vacuum treatment rangefrom about 0.1:1 to about 1,000:1, including about 0.2:1, 0.3:1, 4.0:1,5.0:1, 10.0:1, 20.0:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 500:1, and900:1.

As a result of the processes disclosed above, in some embodiments, thesolvent is substantially removed from the isolated PHA in the PHA-richsolvent phase and the liquid is substantially removed from thePHA-reduced biomass in the PHA-lean liquid phase. In some embodiments,the isolated PHA is dried in a heated vessel to produce substantiallypure isolated PHA (e.g., at least 80% PHA by dry weight, preferably atleast 98% PHA by weight, more preferably at least 99% PHA by weight).

101651 Numerous varieties of heated or drying vessels may be used to drythe isolated PHA, including ovens, centrifugal dryers, air dryers, spraydryers, and freeze dryers, among others. In some embodiments, heat isapplied to a drying vessel to speed the process and/or to remove (e.g.,evaporate traces of solvent from the PHA). It shall be appreciated thatthe moisture content of the isolated PHA will depend, in someembodiments, on the solvent used, and the corresponding separationtechnique used (as described above). For example, a volatile solvent incombination with ultracentrifugation would result in a less moistextracted PHA, while a less active separation technique (e.g., gravityphase separation) would yield a more moist extracted PHA. In someembodiments, internal dryer temperatures range from 20° C. to 40° C. toabout 200° C. In some embodiments, internal temperatures range fromabout 50° C. to 90° C., about 90° C. to 180° C., about 65° C. to 175° C.and overlapping ranges thereof. In some embodiments, outlet temperaturesare substantially lower than inlet on internal temperatures. In someembodiments, outlet temperatures range from 30 ° C. to 90 ° C. In someembodiments, outlet temperatures are between about 35° C. to 40° C.,about 40° C. to 45° C., about 45° C. to 50° C., about 50° C. to 55° C.,about 55 ° C. to90 ° C., and overlapping ranges thereof. It shall alsobe appreciated that the internal and outlet temperatures may be adjustedthroughout the drying process (e.g., the temperature difference mayinitially be large, but decrease over time, or vice versa).

From the above discussion, it shall be appreciated that the type ofdryer used, and the temperatures used (if other than atmospherictemperatures) are easily tailored to correspond to the techniques usedin the extraction process. In some embodiments, particular dryercomponents are beneficial in the isolation of PHA. For example,depending on the moisture content of the extracted PHA (e.g., the amountof residual solvent) particular components of an evaporative-type dryer,such as an oven dryer, rotary dryer, spin flash dryer, spray dryer(equipped with various types of nozzle types, including rotary atomizor,single flow atomizer, mist atomizer, pressure atomizer, dual-flowatomizer) convection heat dryer, tray dryer, scrape-flash dryer, orother dryer type are used. By way of additional example, if a freezedryer (e.g., a lyophilizer) is used, in some embodiments a manifolddryer is used, optionally in conjunction with a heat source. Also by wayof example, a tray lyophilizer can be used, in some embodiments, withthe isolated and dried PHA being stored and sealed in containers (e.g.,vials) before re-exposure to the atmosphere. In certain embodiments,such an approach is used when long-term storage of the PHA is desired.

It shall also be appreciated that certain varieties of heated/dryingapparatuses have adjustable flow rates that can be tailored to themoisture content of the isolated PHA. For example, an isolated PHAhaving a high moisture content would be fed into a dryer at a slowerinput rate to allow a higher degree of drying per unit of PHA inputtedinto the dryer. Conversely, a low moisture content isolated PHA wouldlikely require less time to dry, and therefore could be input at afaster rate. In some embodiments, input rates of isolated PHA range fromseveral hundred liters of isolated PHA-solvent mixture per minute downto several milliliters per minute. For example, input rates can rangefrom about 10 mL/min to about 6 L/min, including about 10 ml/min toabout 50 ml/min, about 50 mL/min to about 100 ml/min, about 100 ml/minto about 500 ml/min, about 500 ml/min to about 1 L/min, about 1 L/min toabout 2 L/min, about 2L/min to about 4 L/min, about 4L/min to about 6L/min, and about 100 L/min to about 500 L/min.

A range of PHA functional characteristics can be attained by mixing onePHA molecule, such as PHB, with various PHA polymers, including PHB. atvarious molecular weights. Therefore, in several embodiments, a firstisolated PHA is heated to reduce the molecular weight of the firstisolated PHA, and then subsequently mixed with a second PHA wherein themolecular weight of the second PHA is higher than the molecular weightof the first PHA. With such embodiments, Applicant has surprisinglydiscovered methods to functionalize one or more PHAs, including PHB. Inadditional embodiments of the invention, the molecular weight of a firstPHA is reduced from about 800,000-5,000,000 Daltons to about 30,000 to800,000 Daltons and mixed with a second PHA with a molecular weight ofabout 800,000 to 5,000,000 Daltons to modify the functionalities of theinput PHAs. In yet another embodiment, a first PHA is mixed with asecond PHA wherein the molecular weight of the first PHA is at least0.1% less than the molecular weight of the second PHA. In someembodiments, the difference in molecular weight between the first andsecond PHA is about 0.1% to 1%, about 1% to 10%, about 10% to 20%, about20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 60%, about60% to 70%, and overlapping ranges thereof. In still additionalembodiments, PHAs having greater differences in molecular weight areused. In yet another embodiment, the molecular weight of a first PHB isreduced to less than about 100,000-500,000 Daltons and mixed with asecond PHA with a molecular weight greater than about 100,000-500,000Daltons to modify the functionality of the input PHB. It shall beappreciated that input PHA and PHB weight may vary from the rangesdisclosed above, but based on the differences in the molecular weights,the alteration in functionality of the input PHB is still achieved.

Purifying the Isolated PHA

In some embodiments, isolated PHA is purified to produce a PHA materialthat is substantially pure PHA. In some embodiments, the isolated PHA ispurified to at least 20% pure PHA by dry weight. In some embodiments,the isolated PHA is purified to at least 55% pure PHA by dry weight,while in some embodiments, the isolated PHA is purified to at least 90%pure PHA by dry weight. In additional embodiments, purity of theisolated PHA is between about 90 and 99.9%, including 91, 92, 93, 94,95, 96, 97, 98, and 99% pure.

In several embodiments, isolated PHA may be recovered by any one, or acombination, of the methods described above, including, but not limitedto: washing, filtration, centrifugation, dewatering, purification,oxidation, non-PHA material removal, solvent removal, and/or drying. Insome embodiments, isolated PHA is recovered according to the manner inwhich it has been removed from the culture. For example, in embodimentsin which solvent-based extraction is employed, a recovery method may beemployed to remove the isolated PHA from the solvent and/or othernon-PHA material. In one embodiment, solvent may be used to extract thePHA, wherein the solvent is then substantially removed from the isolatedPHA by carrying out PHA precipitation and filtration, excess solventdistillation and/or removal, and/or drying, resulting in the recovery ofdry, isolated PHA. In embodiments employing enzyme, surfactant,protonic, hydroxide, and hypochlorite-based extraction techniqueswherein the dissolution of non-PHA material is induced, isolated PHA maybe filtered, washed, separated, centrifuged, and/or dried, resulting inthe recovery of dry, isolated, purified PHA. The resultant PHA, in someembodiments, is further used in downstream processing, includingthermoforming.

Returning PHA-reduced Biomass to the PHA-producing Culture to ConvertPHA-reduced Biomass into PHA

In several embodiments of the invention, the PHA-reduced biomass isreturned to the culture to cause the biomass-utilizing microorganismswithin the culture to convert the carbon within the PHA-reduced biomassinto PHA. By using PHA-reduced biomass as a source of carbon for theproduction of microorganisms in a microorganism fermentation system, aseries of biochemical enzymatic pathways are generated in situ by themicroorganism culture to carry out the metabolic utilization ofPHA-reduced biomass for growth, reproduction, and PHA synthesis.

Without being limited by theory, it is believed that gas-utilizingmicroorganisms and biomass-utilizing microorganisms are able to co-existas a single microorganism consortium because they utilize sources ofcarbon that require distinctly different bioenzymatic assimilationpathways. For instance, while methane metabolism requires the methanemonooxygenase enzyme to catalyze the conversion of methane into methanolfor cellular assimilation, and methane monooxygenase is competitivelyinhibited by a wide range of compounds, it is not inherently deactivatedby high concentrations of cellular biomass, including PHA-reducedbiomass. Similady, the chlorophyll-based metabolic assimilation systemsrequired for the conversion of carbon dioxide into biomass and PHA arenot inherently deactivated or competitively inhibited by highconcentrations of cellular biomass, including PHA-reduced biomass.Likewise, the enzymatic architecture enabling the metabolic utilization,breakdown, and/or assimilation of PHA-reduced biomass is not inherentlydeactivated or competitively inhibited by high concentrations of methaneand/or carbon dioxide, particularly as the process requires neithermethane monooxygenase nor chlorophyll. Without being limited by theory,Applicant believes that the relatively non-competitive, and in somecases commensal or mutualistic relationships between microorganismsconsuming a carbon-containing gas and a PHA-reduced biomass, make itpossible to create a microorganism culture comprising biomass-utilizingmicroorganisms and gas-utilizing microorganisms, wherein bothcarbon-containing gases and PHA-reduced biomass may be metabolized assimultaneously assimilable sources of carbon.

In the case of autotrophic, methanotrophic, and/or biomass-utilizingmicroorganisms, Applicant has found that a mutualistic,positive-feedback loop relationship can be created in a single (oroptionally multiple) reaction vessel. In such embodiments, the oxygencreated by autotrophic metabolism is utilized by methanotrophic and/orbiomass-utilizing microorganisms for metabolic functions, the carbondioxide created by methanotrophic and/or biomass-utilizing microorganismmetabolism is utilized for autotrophic metabolism, the methane and/orcarbon dioxide created by anaerobic methanogenic microorganisms isutilized by methanotrophic microorganisms, and the biomass created bymethanotrophic, autotrophic, and/or heterotrophic microorganisms is usedto provide a source of carbon to methanogenic and/or other heterotrophicmicroorganisms. Due to the microscopic-level induction of oxygen and/orcarbon dioxide created therein, mass transfer efficiencies in severalembodiments are significantly improved over traditional gas inductionmeans, such as gas sparging, mechanical mixing, static mixing, or othermeans known in the art. To applicant's knowledge, prior to thedisclosure herein, the use of autotrophic microorganisms cultured inassociation with heterotrophic microorganisms has never been suggestedas a means to improve mass transfer efficiencies, supply oxygen, and/oraugment microorganism growth rates in a positive feedback loop system.

In several further embodiments of the invention, PHA-reduced biomass isused by heterotrophic microorganisms, including acidogenic, acetogenic,and methanogenic microorganisms, to produce methane, which is furtherutilized by methanotrophic microorganisms to produce biomass, includingPHA. in some embodiments of the invention, anaerobic microorganismscoexist with aerobic microorganisms under microaerobic conditions (e.g.,mean dissolved oxygen concentrations approximately 0.00-1.0 ppm,including about 0.001 to 0.002 ppm, 0.002 to 0.03 ppm, 0.03 to 0.04 ppm,0.04 to 0.5 ppm, 0.5 to 0.6 ppm, 0.6 to 0.7 ppm, 0.7 to 0.8 ppm, 0.8 to0.9 ppm, 0.9 to 1.0 ppm, and overlapping ranges thereof.

In some embodiments of the invention, heterotrophic, methanotrophic,methanogenic, and/or autotrophic microorganisms are divided intomultiple stages and vessels, in particular, anaerobic and aerobicstages, in order to carry out the conversion of PHA-reduced biomass intomethane and PHA. In further embodiments of the invention, PHA-reducedbiomass is returned to the culture using one or more vessels, whereby itis first converted to carbon dioxide, methane, and/or volatile organiccompounds by a heterotrophic, e.g., methanogenic, microorganismconsortium under anaerobic conditions and then converted to PHA bymethanotrophic microorganisms under aerobic conditions, whereby carbondioxide is also metabolized or otherwise used by autotrophicmicroorganisms, methanotrophic microorganisms, and heterotrophicmicroorganisms.

In several embodiments, light intensity is utilized to regulate thegrowth rate of heterotrophic and/or methanotrophic microorganisms. Insome embodiments, light intensity is manipulated to regulate thegeneration of oxygen by autotrophic microorganisms. In some embodiments,the rate of oxygen generated by autotrophic microorganisms issubsequently used to control the growth and metabolism of heterotrophicand methanotrophic microorganisms.

In several embodiments, carbon dioxide is supplied to autotrophicmicroorganisms in the form of carbon dioxide created by methanotrophicand/or heterotrophic microorganisms. In some embodiments, each of thesevarieties of microorganism is cultured in a single vessel. In someembodiments, methane, sugar, biomass, and/or another non-carbon dioxidesource of carbon is used to grow autotrophic microorganisms. Toapplicant's knowledge, autotrophic microorganisms have never beencultured using methane as a sole carbon input.

Some gas-utilizing microorganisms are unable to produce highconcentrations of intracellular PHA. However, according to severalembodiments, certain microorganism consortiums utilizing PHA-reducedbiomass, or derivatives thereof, as a source of carbon are able togenerate high intracellular PHA concentrations and thus effectivelyconvert low PHA concentration biomass derived from a carbon-containinggas into a high PHA concentration biomass material under the conditionsdisclosed herein. In several embodiments, the concentration (by weight)of intracellular PHA is between about 10% to 30%, 30% to 50%, 50% to70%, 70% to 80%, 80% to 90%, 90% or more, and overlapping rangesthereof. Thus, in one embodiment, the culture is contacted with thePHA-reduced biomass and then manipulated, according to the processesdescribed herein, to effect PHA synthesis, wherein the PHA-reducedbiomass is converted into PHA by biomass-utilizing microorganisms. Insome embodiments, PHA synthesis is induced by nutrient limitation,nutrient excess, nutrient imbalance, or large shifts in nutrientconcentration. In still further embodiments, PHA synthesis is induced byreducing the availability of nitrogen, oxygen, phosphorus, or magnesiumto the culture. In some embodiments, these nutrients are simultaneouslyreduced (to varying or similar degrees). In some embodiments, thenutrients are reduced sequentially. In some embodiments, only one of thenutrients is reduced. For example, in certain embodiments, PHA synthesisis induced by reducing the availability of oxygen to the culture. Insome embodiments, this is achieved by manipulating the flow rate of airor oxygen into the growth medium. In some embodiments, manipulation ofthe flow rate of other carbon-containing gases, such as methane and/orcarbon dioxide, into the growth medium, or otherwise manipulating therate of gas transfer in a system (e.g., by adjusting mixing rates orlight injection rates) is employed. In one embodiment, oxygen limitationis induced by reducing the flow rate of oxygen into the growth medium.In another embodiment, oxygen limitation is induced by reducing the rateof light transmission into the medium to reduce the production of oxygenby autotrophic microorganisms. In some embodiments of the invention, theconcentration of PHA generated in a biomass-utilizing microorganismculture utilizing PHA-reduced biomass as a source of carbon is at least5%, at least 20%, or at least 50% by dry cell weight; in particularlypreferred embodiments of the invention, the concentration of PHA in abiomass-utilizing microorganism is at least 80% by dry cell weight.

In some embodiments, a PHA-reduced biomass recycling system is utilizedwherein substantially all (e.g., at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 98%) of the PHA-reducedbiomass produced is contacted with the culture until it is convertedinto PHA. In some such embodiments, solid sources of carbon aresubstantially output from the process or culture only in the form ofisolated PHA.

In several embodiments, as carbon-containing gases are continually addedto the medium to effect the production of biomass, the process disclosedabove is repeated. Specifically, as the process continues, a portion ofthe PHA-containing biomass from the culture is removed from the medium,PHA is extracted from the PHA-containing biomass, PHA-reduced biomass isseparated from isolated PHA, isolated PHA is recovered, purified, anddried, and PHA-reduced biomass is sent back to the culture and convertedby the culture into PHA. In one embodiment, substantially allPHA-reduced biomass produced is contacted with the culture until it isconverted into isolated PHA, and solid sources of carbon aresubstantially output from the process only in the form of isolated PHA.In other embodiments, carbon is substantially output from the systemonly in the form of PHA and methane, carbon dioxide, and/or volatileorganic compounds.

The following example is provided to further illustrate certainembodiments within the scope of the invention. The example is not to beconstrued as a limitation of any embodiments, since numerousmodifications and variations are possible without departing from thespirit and scope of the invention.

EXAMPLE 1

A fermentation system comprising one or more vessels are partiallyfilled with one or more liquid growth mediums, wherein the mediumcomprises methanotrophic, autotrophic, methanotrophic, and/or otherheterotrophic or biomass-utilizing microorganisms containing PHA, and,per liter of water, 0.7-1.5 g KH₂PO₄, 0.7-1.5 g K₂HPO₄, 0.7-1.5 g KNO₃,0.7-1.5 g NaCl, 0.1-0.3 g MgSO₄, 24-28 mg CaCl₂*2H₂O, 5.0-5.4 mg EDTANa₄(H₂O)₂, 1.3-1.7 mg FeCl₂*4H₂O, 0.10-0.14 mg CoCl₂*6H₂O, 0.08-1.12 mgMDCl₂*2H₂O, 0.06-0.08 mg ZnCl₂, 0.05-0.07 mg H₃BO₃, 0.023-0.027 mgNiCl₂*6H₂O, 0.023-0.027 mg NaMoO₄*2H₂O, 0.011-0.019 mg CuCl₂*2H₂O. Oneor more of the mediums are anaerobic and/or aerobic, and carboncontaining gases, including methane, carbon dioxide, and volatileorganic compounds, as well as optionally air or oxygen, are fed into allor part of the system to induce the growth and reproduction ofmicroorganisms through the utilization of carbon-containing gases, aswell as the production of PHA.

Next, a portion of the media volume of the fermentation system is passedthrough a basket centrifuge to increase the solids content of the mediumto approximately 167 gIL. The solids-containing centrate phase of thecentrifuged solution is then transferred to a PHA extraction vessel, andthe substantially solids-free filtrate phase of the centrifuged solutionis recycled back to the fermentation system.

In some embodiments, the solids-containing centrate phase is optionallychemically pre-treated prior to extraction. In some embodiments, one ormore of acids, bases, chloride, ozone, and hydrogen peroxide is added.In several embodiments, chemical pre-treatment increases the efficiencyand yield of the subsequent extraction process. In some embodiments, thechemical pre-treatment functions to break down the cell well (partiallyor fully), thereby liberating a greater portion of the intracellularPHA. In some embodiments, chemical pre-treatment dissolves and/orremoves impurities that negatively impact the PHA extraction process. Insome embodiments, chemical pre-treatment enhances cell agglomeration,which increases the percentage of microorganisms that are extracted(e.g., cells in an agglomerated mass are not separated or lost intransfer steps). Next, following optional chemical pre-treatment,solvent is added into the PHA extraction vessel to create a solventsolution, and the solvent solution is then mixed for a period of time tocause the PHA in both the microorganisms to dissolve into the solvent,and thereby create PHA-rich solvent and PHA-reduced biomass. Over thecourse of a defined mixing period (e.g., 0.1-10 hours), the PHA contentof the microorganisms is reduced by about 80% as it is dissolved intothe solvent.

Next, the solvent solution comprising the PHA-rich solvent andPHA-reduced biomass is passed through a filter located at the bottom ofthe PHA extraction vessel, and the PHA-rich solvent is thereby separatedfrom the PHA-reduced biomass. Water is then added to the PHA extractionvessel to create a water-biomass solution, and the water-biomasssolution is then heated to 75° C. to cause any remaining solventassociated with the PHA-reduced biomass to exit the PHA extractionvessel as a gaseous vapor. The vapor discharged from the PHA extractionvessel is then passed through a heat exchanger and recovered as liquidsolvent. Meanwhile, the PHA-rich solvent is transferred to a PHApurification vessel and mixed with room temperature water to create awater-solvent solution. The water-solvent solution is then heated tocause i) the solvent to exit the PHA extraction vessel as a gaseousvapor and ii) the isolated PHA to precipitate into the water and/orbecome a solid. The solvent vapor created by heating the water-solventsolution is then passed through a heat exchanger and converted intoliquid solvent.

The isolated PHA is then substantially dewatered by filtration in aNutsche filter, and the Nutsche filter containing the substantiallydewatered isolated PHA is then heated to remove any additional volatilecompounds, including trace water and/or solvent. Following heat dryingin the Nutsche filter, the isolated PHA is recovered as substantiallypure PHA (e.g., greater than about 90% PHA).

Concurrently, the water-biomass solution comprising PHA-reduced biomassand water is transferred from the PHA extraction vessel back into thefermentation system, where the PHA-reduced biomass is contacted with oneor more of the microorganism cultures. Next, the medium of thefermentation system is manipulated to cause the one or moremicroorganisms within the system to metabolize the PHA-reduced biomassas a source of carbon and nutrients.

Depending on the embodiment, the culture conditions are adjusted todetermine the point at which the inception of the growth or PHAmetabolism phase occurs. As discussed herein, manipulation of theconcentration of one or more growth culture media nutrients can alterthe metabolic pathways favored by certain microorganisms. Additionally,the use of PHA-reduced biomass-derived carbon for the production ofadditional biomass versus the production of PHA can be tailored based onwhether the intent is to grow the culture (e.g., increase the overallbiomass) or to harvest PHA (e.g., shift the culture from growth phase toproduction of PHA). As such, the reduction, increase, or adjustment ofthe concentration certain growth nutrients, and the timing of suchadjustment, plays a role in the metabolic state and PHA production ofthe culture. Adjustment of growth nutrients can occur at any point afterthe PHA-reduced biomass is returned to the microorganism system. In someembodiments, adjustment is immediate (e.g., within minutes to a fewhours). In some embodiments, a longer period of time elapses. In someembodiments, adjustment in one or more growth nutrients occurs afterabout 2 to 4 hours, after about 4 to 6 hours, after about 6 to 8 hours,after about 8 to 10 hours, after about 10 to 12 hours, after about 12 to14 hours, after about 14 to 18 hours, after about 18 to 24 hours, andoverlapping ranges thereof. In still additional embodiments, adjustmentin one or more growth nutrients occurs after about 2 to 5 days, 5 to 10days, 10 to 15 days, 15 to 20 days, 20 to 30 days, 30 to 50 days, andoverlapping ranges thereof. In some embodiments, longer times elapseprior to adjusting one or more growth nutrients to induce PHApolymerization.

After a desired period of time has elapsed, the dissolved oxygen and/ornitrogen concentration (or concentration of another nutrient) of one ormore parts of the medium is reduced or adjusted to cause one or more ofthe microorganisms within the system to utilize the PHA-reduced biomassin the medium as a source of carbon for the synthesis of PHA. In someembodiments, the percent adjustment ranges from about 20% to about 100%,including 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%,70% to 80%, 80% to 90%, 90% to 100%, and overlapping ranges thereof. Itshall be appreciated that, depending on the characteristics of a culturein a given embodiment, a specific percentage reduction, increase, oradjustment in nutrient may not be necessary, but a reduction, increase,or adjustment is used that is sufficient to convert certain cells from arelative growth phase to a relative PHA synthesis phase. Afterapproximately 12-24 hours of PHA synthesis, substantially all of thePHA-reduced biomass within the growth medium has been metabolized intobiomass-utilizing microorganism biomass, including PHA. It shall beappreciated that, in certain embodiments, greater or lesser PHAsynthesis times result in varying percentages of the PHA-reduced biomasswithin the growth medium being metabolized into biomass, including PHA.

As carbon containing gases are continually added to the fermentationsystem to effect the production of biomass, the process is repeated,wherein solid sources of carbon substantially exit the system only inthe form of PHA. Specifically, as the process continues, a portion ofthe PHA-containing biomass from the fermentation vessel is passedthrough a dewatering centrifuge to increase the solids content of thePHA-containing biomass, PHA is extracted from the removed PHA-containingbiomass using a solvent-based extraction system to create PHA-reducedbiomass and isolated PHA, PHA-reduced biomass is separated from isolatedPHA, isolated PHA is recovered, purified, and dried, and PHA-reducedbiomass is sent back to the fermentation system and converted bymicroorganisms into PHA, such that substantially all PHA-reduced biomassproduced is contacted with the culture until it is converted intoisolated PHA, and wherein solid sources of carbon are substantiallyoutput from the process only in the form of isolated PHA.

While the above description of several compositions, systems, andmethods contains many specificities, it should be understood that theembodiments of the present invention described above are illustrativeonly and are not intended to limit the scope of the invention. Numerousand various modifications can be made without departing from the spiritof the embodiments described herein. Accordingly, the scope of theinvention should not be solely determined by the embodiments describedherein, but also by the appended claims and their legal equivalents.

1.-102. (canceled)
 103. A process for the production of apolyhydroxyalkanoate (PHA), comprising the steps of: (a) providing amicroorganism culture comprising PHA-containing biomass; (b) removing aportion of said PHA-containing biomass from said culture; (c) increasingthe concentration of PHA in said removed PHA-containing biomass toproduce PHA-rich biomass and PHA-reduced biomass; and (d) returning saidPHA-reduced biomass to said culture to cause said PHA-reduced biomass tobe used to produce PHA.
 104. The process of claim 103, wherein saidculture converts the carbon within said PHA-reduced biomass into PHA.105. The process of claim 104, wherein at least a portion of said carbonis non-PHA carbon.
 106. The process of claim 104, wherein said carbon ismetabolized by said culture to produce converted biomass, wherein saidconverted biomass is used by said culture to produce PHA.
 107. Theprocess of claim 104, wherein said converted biomass is carbon dioxideand/or methane.
 108. The process of claim 104, wherein said convertedbiomass is metabolically generated material, a volatile fatty acid, avolatile organic compound, a carbon-containing compound, intracellular,cellular, and/or extracellular material, a polymer, an amino acid, anucleic acid, a carbohydrate, a lipid, a sugar, a polyhydroxyalkanoate,a chemical, and/or a metabolic derivative, intermediary, and/orend-product.
 109. The process of claim 103, wherein said PHA-reducedbiomass metabolically synthesizes PHA.
 110. The process of claim 109,wherein said PHA-reduced biomass metabolically synthesizes PHA byconverting a carbon-containing gas into PHA.
 111. The process accordingto claim 103, wherein said microorganism culture comprisesmethanotrophic microorganisms, carbon-dioxide utilizing microorganisms,heterotrophic microorganisms, autotrophic microorganisms, cyanobacteria,biomass-utilizing microorganisms, methanogenic microorganisms, aerobicmicroorganisms, anaerobic microorganisms, acidogenic microorganisms,and/or acetogenic microorganisms, wherein said microorganism are capableof using carbon-containing gases and/or PHA-reduced biomass as a sourceof carbon for metabolism.
 112. The process according to claim 103,wherein increasing the concentration of said PHA in said removedPHA-containing biomass comprises reducing the concentration of water insaid removed PHA-containing biomass, and wherein said PHA-rich biomasscomprises said removed PHA-containing biomass wherein the waterconcentration has been reduced, and wherein said PHA-reduced biomasscomprises said removed PHA-containing biomass wherein the waterconcentration has not been reduced.
 113. The process according to claim103, wherein increasing the concentration of PHA in said removedPHA-containing biomass comprises extracting said PHA from said removedPHA-containing biomass.
 114. The process of claim 113, where extractingsaid PHA from said removed PHA-containing biomass comprises mixing saidPHA-containing biomass with an extraction agent or mechanism selectedfrom the group consisting of solvents, solvent washing, chemicaltreatment, microwave treatment, simple or fractional distillation,supercritical carbon dioxide, heat, enzymes, surfactants, acids, bases,hypochlorite, peroxides, polymers, bleaches, ozone, EDTA, and/or acombination thereof, and said extraction is optionally performed with asolvent selected from the group consisting of methylene chloride,acetone, ethanol, methanol, ketones, alcohol, chloroform,dichloroethane, water, and/or carbon dioxide, and wherein saidextraction is optionally performed by mixing said removed PHA-containingbiomass with a mechanism selected from the group consisting ofsonication, homogenization, distillation, spray drying, hypochloritenon-PHA dissolution, protonic non-PHA dissolution, non-PHA dissolution,enzymatic treatment, and/or freeze drying.
 115. The process of claim103, wherein natural and/or artificial light is utilized to influencethe metabolism of said culture.
 116. The process according to claim 103,wherein said culture uses at least a portion of carbon-containing gas toproduce said PHA, wherein said gas is derived from one or more sourcesfrom the group consisting of: landfills, wastewater treatment plants,power production facilities or equipment, agricultural digesters, oilrefineries, natural gas refineries, natural gas streams, cementproduction facilities, and/or anaerobic organic waste digesters. 117.The process according to claim 103, wherein said culture uses at least aportion of carbon-containing gas to produce said PHA, wherein said gasis derived from one or more sources from the group consisting of:landfill, wastewater treatment plant, power production facility, oranaerobic decomposition source.
 118. The process according to claim 103,wherein the metabolism, growth, reproduction, and/or PHA synthesis ofsaid culture is controlled, manipulated, and/or affected by a growthmedium, wherein said medium comprises nutrients, wherein said nutrientscomprise at least one or more of the following: water, nitrogen,methane, carbon dioxide, phosphorus, oxygen, magnesium, potassium, iron,copper, sulfur, manganese, molybdenum, calcium, chlorine, boron, zinc,aluminum, nickel, and/or sodium.
 119. The process of claim 118, whereinsaid production of said PHA is caused by increasing, reducing, ormaintaining the concentration of one or more nutrient within saidmedium.
 120. The process of claim 118, wherein said culture is caused togrow and/or reproduce by increasing, reducing, or maintaining theconcentration of one or more nutrient within said medium.
 121. Theprocess of claim 118, wherein said medium is controlled to cause saidculture to undergo a period of growth and reproduction, wherein saidmedium is subsequently controlled to cause said culture to undergo aperiod of PHA synthesis, wherein said medium is subsequently controlledto cause said culture to undergo a period of growth and reproduction,wherein microorganisms within said culture that reproduce moreefficiently as a result of higher intracellular concentrations of PHAare caused to be produced in higher concentrations than microorganismswithin said culture that reproduce less efficiently as a result of lowerintracellular concentrations of PHA.
 122. The process of claim 121,wherein said microorganisms reproduce more efficiently as a result ofhigher intracellular concentrations of PHA by using said intracellularPHA as a source of carbon, energy, power, or reducing power.
 123. Theprocess of claim 121, wherein the shift from said reproduction period tosaid polymerization period to said reproduction periods is consecutivefor at least part of the culture and does not comprise a period whereinno carbon or substantially no carbon is available for metabolism by saidculture or wherein the carbon available to said culture is significantlyreduced.
 124. A polyhydroxyalkanoate material comprising carbon derivedfrom carbon-containing gas and PHA-reduced biomass.
 125. A process forthe production of a polyhydroxyalkanoate (PHA), comprising the steps of:(a) providing a microorganism culture, (b) providing a culture mediumcomprising nutrients, wherein said medium influences the metabolism,growth, reproduction, and/or PHA synthesis of said culture, (c)providing a source of carbon that can be metabolized by said culture,(d) combining said culture, said medium, and said carbon, (e)controlling said medium to cause said culture to use said carbon toreproduce, (f) controlling said medium to cause said culture to use saidcarbon to synthesize intracellular PHA, (g) controlling said medium tocause said culture to use said carbon to reproduce, whereinmicroorganisms within said culture that reproduce more efficiently as aresult of higher intracellular concentrations of PHA are produced inhigher concentrations than microorganisms within said culture thatreproduce less efficiently as a result of lower intracellularconcentrations of PHA.
 126. The process of claim 125, wherein at least aportion of said culture is removed between step (e) and (f).
 127. Theprocess of claim 125, wherein at least a portion of said culture isremoved between step (f) and (g).
 128. The process of claim 125, whereinat least a portion of said culture is removed between step (e) and (g).129. The process of claim 125, wherein said culture is exposed to asource of microorganisms that were not previously present in saidculture.
 130. The process of claim 125, wherein at least a portion ofsaid culture is removed from said culture, wherein the concentration ofPHA in said removed culture is increased to produce a PHA-rich biomassand non-PHA-rich biomass, wherein said non-PHA-rich biomass is returnedto said non-removed culture and used as a source of carbon to producePHA.